LIPID COMPRISING POLYUNSATURATED FATTY ACIDS

Abstract

The present invention relates to extracted plant lipid, comprising fatty acids in an esterified form.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Patent Application No. 61/782,680, filed Mar. 14, 2013, U.S. Provisional Patent Application No. 61/697,676, filed Sep. 6, 2012, U.S. Provisional Patent Application No. 61/663,344, filed Jun. 22, 2012, and U.S. Provisional Patent Application No. 61/660,392, filed Jun. 15, 2012, the entire contents of each of which are hereby incorporated by reference into the subject application.

REFERENCE TO SEQUENCE LISTING

[0002] This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named "130614_--2251_--84199_A_Sequence_Listing_REB.txt," which is 369 kilobytes in size, and which was created Jun. 14, 2013 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Jun. 14, 2013 as part of this application.

FIELD OF THE INVENTION

[0003] The present invention relates to extracted plant lipid, comprising fatty acids in an esterified form.

BACKGROUND OF THE INVENTION

[0004] Omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) are now widely recognized as important compounds for human and animal health. These fatty acids may be obtained from dietary sources or by conversion of linoleic (LA, 18:2ω6) or α-linolenic (ALA, 18:3ω3) fatty acids, both of which are regarded as essential fatty acids in the human diet. While humans and many other vertebrate animals are able to convert LA or ALA, obtained from plant sources to C22 they carry out this conversion at a very low rate. Moreover, most modern societies have imbalanced diets in which at least 90% of polyunsaturated fatty acids (PUFA) are of the ω6 fatty acids, instead of the 4:1 ratio or less for ω6:ω3 fatty acids that is regarded as ideal (Trautwein, 2001). The immediate dietary source of LC-PUFAs such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3) for humans is mostly from fish or fish oil. Health professionals have therefore recommended the regular inclusion of fish containing significant levels of LC-PUFA into the human diet. Increasingly, fish-derived LC-PUFA oils are being incorporated into food products and in infant formula, for example. However, due to a decline in global and national fisheries, alternative sources of these beneficial health-enhancing oils are needed.

[0005] Flowering plants, in contrast to animals, lack the capacity to synthesise polyunsaturated fatty acids with chain lengths longer than 18 carbons. In particular, crop and horticultural plants along with other angiosperms do not have the enzymes needed to synthesize the longer chain ω3 fatty acids such as EPA, docosapentaenoic acid (DPA, 22:5ω3) and DHA that are derived from ALA. An important goal in plant biotechnology is therefore the engineering of crop plants which produce substantial quantities of LC-PUFA, thus providing an alternative source of these compounds.

LC-PUFA Biosynthesis Pathways

[0006] Biosynthesis of LC-PUFAs in organisms such as microalgae, mosses and fungi usually occurs as a series of oxygen-dependent desaturation and elongation reactions (FIG. 1). The most common pathway that produces EPA in these organisms includes a Δ6-desaturation, Δ6-elongation and Δ5-desaturation (termed the Δ6-desaturation pathway) whilst a less common pathway uses a Δ9-elongation, Δ8-desaturation and Δ5-desaturation (termed the Δ9-desaturation pathway). These consecutive desaturation and elongation reactions can begin with either the ω6 fatty acid substrate LA, shown schematically as the upper left part of FIG. 1 (ω6) or the ω3 substrate ALA through to EPA, shown as the lower right part of FIG. 1 (ω3). If the initial Δ6-desaturation is performed on the ω6 substrate LA, the LC-PUFA product of the series of three enzymes will be the ω6 fatty acid ARA. LC-PUFA synthesising organisms may convert ω6 fatty acids to ω3 fatty acids using an ω3-desaturase, shown as the Δ17-desaturase step in FIG. 1 for conversion of arachidonic acid (ARA, 20:4ω6) to EPA. Some members of the ω3-desaturase family can act on a variety of substrates ranging from LA to ARA. Plant ω3-desaturases often specifically catalyse the Δ15-desaturation of LA to ALA, while fungal and yeast ω3-desaturases may be specific for the Δ17-desaturation of ARA to EPA (Pereira et al., 2004a; Zank et al., 2005). Some reports suggest that non-specific ω3-desaturases may exist which can convert a wide variety of ω6 substrates to their corresponding ω3 products (Zhang et al., 2008).

[0007] The conversion of EPA to DHA in these organisms occurs by a Δ5-elongation of EPA to produce DPA, followed by a Δ4-desaturation to produce DHA (FIG. 1). In contrast, mammals use the so-called "Sprecher" pathway which converts DPA to DHA by three separate reactions that are independent of a Δ4-desaturase (Sprecher et al., 1995).

[0008] The front-end desaturases generally found in plants, mosses, microalgae, and lower animals such as Caenorhabditis elegans predominantly accept fatty acid substrates esterified to the sn-2 position of a phosphatidylcholine (PC) substrate. These desaturases are therefore known as acyl-PC, lipid-linked, front-end desaturases (Domergue et al., 2003). In contrast, higher animal front-end desaturases generally accept acyl-CoA substrates where the fatty acid substrate is linked to CoA rather than PC (Domergue et al., 2005). Some microalgal desaturases and one plant desaturase are known to use fatty acid substrates esterified to CoA (Table 2).

[0009] Each PUFA elongation reaction consists of four steps catalysed by a multi-component protein complex: first, a condensation reaction results in the addition of a 2C unit from malonyl-CoA to the fatty acid, resulting in the formation of a β-ketoacyl intermediate. This is then reduced by NADPH, followed by a dehydration to yield an enoyl intermediate. This intermediate is finally reduced a second time to produce the elongated fatty acid. It is generally thought that the condensation step of these four reactions is substrate specific whilst the other steps are not. In practice, this means that native plant elongation machinery is capable of elongating PUFA providing that the condensation enzyme (typically called an `elongase`) specific to the PUFA is introduced, although the efficiency of the native plant elongation machinery in elongating the non-native PUFA substrates may be low. In 2007 the identification and characterisation of the yeast elongation cycle dehydratase was published (Denic and Weissman, 2007).

[0010] PUFA desaturation in plants, mosses and microalgae naturally occurs to fatty acid substrates predominantly in the acyl-PC pool whilst elongation occurs to substrates in the acyl-CoA pool. Transfer of fatty acids from acyl-PC molecules to a CoA carrier is performed by phospholipases (PLAs) whilst the transfer of acyl-CoA fatty acids to a PC carrier is performed by lysophosphatidyl-choline acyltransferases (LPCATs) (FIG. 21) (Singh et al., 2005).

Engineered production of LC-PUFA

[0011] Most LC-PUFA metabolic engineering has been performed using the aerobic Δ6-desaturation/elongation pathway. The biosynthesis of γ-linolenic acid (GLA, 18:3ω6) in tobacco was first reported in 1996 using a Δ6-desaturase from the cyanobacterium Synechocystis (Reddy and Thomas, 1996). More recently, GLA has been produced in crop plants such as safflower (73% GLA in seedoil; Knauf et al., 2006) and soybean (28% GLA; Sato et al., 2004). The production of LC-PUFA such as EPA and DHA involves more complicated engineering due to the increased number of desaturation and elongation steps involved. EPA production in a land plant was first reported by Qi et al. (2004) who introduced genes encoding a Δ9-elongase from Isochrysis galbana, a Δ8-desaturase from Euglena gracilis and a Δ5-desaturase from Mortierella alpina into Arabidopsis yielding up to 3% EPA. This work was followed by Abbadi et al. (2004) who reported the production of up to 0.8% EPA in flax seed using genes encoding a Δ6-desaturase and Δ6-elongase from Physcomitrella patens and a Δ5-desaturase from Phaeodactylum tricornutum.

[0012] The first report of DHA production, and to date the highest levels of VLC-PUFA production reported, was in WO 04/017467 where the production of 3% DHA in soybean embryos is described, but not seed, by introducing genes encoding the Saprolegnia diclina Δ6-desaturase, Mortierella alpina Δ6-desaturase, Mortierella alpina Δ5-desaturase, Saprolegnia diclina Δ4-desaturase, Saprolegnia diclina Δ17-desaturase, Mortierella alpina Δ6-elongase and Pavlova lutheri Δ5-elongase. The maximal EPA level in embryos also producing DHA was 19.6%, indicating that the efficiency of conversion of EPA to DHA was poor (WO 2004/071467). This finding was similar to that published by Robert et al. (2005), where the flux from EPA to DHA was low, with the production of 3% EPA and 0.5% DHA in Arabidopsis using the Danio rerio Δ5/6-desaturase, the Caenorhabditis elegans Δ6-elongase, and the Pavlova salina Δ5-elongase and Δ4-desaturase. Also in 2005, Wu et al. published the production of 25% ARA, 15% EPA, and 1.5% DHA in Brassica juncea using the Pythium irregulare Δ6-desaturase, a Thraustochytrid Δ5-desaturase, the Physcomitrella patens Δ6-elongase, the Calendula officianalis Δ12-desaturase, a Thraustochytrid Δ5-elongase, the Phytophthora infestans Δ17-desaturase, the Oncorhyncus mykiss LC-PUFA elongase, a Thraustochytrid Δ4-desaturase and a Thraustochytrid LPCAT (Wu et al., 2005). Summaries of efforts to produce oil-seed crops which synthesize ω3 LC-PUFAs is provided in Venegas-Caleron et al. (2010) and Ruiz-Lopez et al. (2012). As indicated by Ruiz-Lopez et al. (2012), results obtained to date for the production of DHA in transgenic plants has been no where near the levels seen in fish oils.

[0013] There therefore remains a need for more efficient production of LC-PUFA in recombinant cells, in particular of DHA in seeds of oilseed plants.

SUMMARY OF THE INVENTION

[0014] The present inventors have identified methods and plants for producing lipid with high levels of DHA.

[0015] In a first aspect, the present invention provides extracted plant lipid, comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA in the total fatty acid content of the extracted lipid is about 7% to 20%.

[0016] In an embodiment, the extracted lipid has one or more or all of the following features

[0017] i) the level of palmitic acid in the total fatty acid content of the extracted lipid is between about 2% and 18%, between about 2% and 16%, or between about 2% and 15%,

[0018] ii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than about 6%, less than about 3%, less than about 2%, or less than about 1%,

[0019] iii) the level of oleic acid in the total fatty acid content of the extracted lipid is between about 1% and about 30%, between about 3% and about 30%, between about 6% and about 30%, between 1% and about 20%, between about 30% and about 60%, between about 45% to about 60%, or is about 30%,

[0020] iv) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between about 4% and about 35%, between about 4% and about 20%, or between about 4% and 17%,

[0021] v) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between about 4% and about 40%, between about 7% and about 40%, between about 10% and about 35%, between about 20% and about 35%, between about 4% and about 16%, or between about 2% and about 16%,

[0022] vi) the level of γ-linolenic acid (GLA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, between 0.05% and about 7%, between 0.05% and about 4%, between 0.05% and about 3%, or between 0.05% and about 2%,

[0023] vii) the level of stearidonic acid (SDA) in the total fatty acid content of the extracted lipid is less than about 7%, less than about 6%, less than about 4%, less than about 3%, between about 0.05% and about 7%, between about 0.05% and about 6%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between 0.05% and about 2%,

[0024] viii) the level of eicosatetraenoic acid (ETA) in the total fatty acid content of the extracted lipid is less than about 6%, less than about 5%, less than about 4%, less than about 1%, less than about 0.5%, between about 0.05% and about 6%, between about 0.05% and about 5%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between about 0.05% and about 2%,

[0025] ix) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 2%, less than about 1%, between about 0.05% and about 4%, between about 0.05% and about 3%, between about 0.05% and about 2%, or between about 0.05% and about 1%,

[0026] x) the level of eicosapentaenoic acid (EPA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, less than about 2%, between about 0.05% and about 10%, between about 0.05% and about 5%, between about 0.05% and about 3%, or between about 0.05% and about 2%,

[0027] xi) the level of docosapentaenoic acid (DPA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, less than about 2%, between about 0.05% and about 8%, between about 0.05% and about 5%, between about 0.05% and about 3%, or between about 0.05% and about 2%,

[0028] xii) the level of DHA in the total fatty acid content of the extracted lipid is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, between about 8% and 20%, between about 10% and 20%, between about 11% and 20%, between about 10% and about 16%, or between about 14% and 20%,

[0029] xiii) the lipid comprises ω6-docosapentaenoic acid (22:5.sup.Δ4, 7, 10, 13, 16) in its fatty acid content,

[0030] xiv) the lipid is essentially free of ω6-docosapentaenoic acid (22:5.sup.Δ4, 7, 10, 13, 16) in its fatty acid content,

[0031] xv) the lipid is essentially free of SDA, EPA and ETA in its fatty acid content,

[0032] xvi) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%, between about 4% and about 20%, between about 6% and about 20%, between about 4% and about 60%, between about 30% and about 60%, or between about 45% and about 60%,

[0033] xvii) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, between about 8% and about 25%, or between 8% and about 22%,

[0034] xviii) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, between about 50% and about 75%, or between about 60% and about 75%,

[0035] xix) the level of total ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 35% and about 50%, between about 20% and about 35%, between about 6% and 20%, less than about 20%, less than about 16%, less than about 10%, between about 1% and about 16%, between about 2% and about 10%, or between about 4% and about 10%,

[0036] xx) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is less than about 10%, less than about 8%, less than about 6%, less than 4%, between about 1% and about 20%, between about 1% and about 10%, between about 0.5% and about 8%, or between about 0.5% and 4%.

[0037] xxi) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between 36% and about 65%, between about 40% and about 60%, between about 20% and about 35%, between about 10% and about 20%, about 25%, about 30%, about 35% or about 40%,

[0038] xxii) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 9% and about 33%, between about 10% and about 20%, between about 20% and about 30%, between about 12% and about 25%, about 13%, about 15%, about 17% or about 20%,

[0039] xxiii) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 1.0 and about 3.0, between about 0.1 and about 1, between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 1.0, about 0.1 or about 0.2,

[0040] xxiv) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 1.0 and about 3.0, between about 0.1 and about 1, between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 0.1, about 0.2 or about 1.0,

[0041] xxv) the fatty acid composition of the lipid is based on an efficiency of conversion of oleic acid to LA by Δ12-desaturase of at least about 60%, at least about 70%, at least about 80%, between about 60% and about 98%, between about 70% and about 95%, or between about 75% and about 90%,

[0042] xxvi) the fatty acid composition of the lipid is based on an efficiency of conversion of ALA to SDA by Δ6-desaturase of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, between about 30% and about 70%, between about 35% and about 60%, or between about 50% and about 70%,

[0043] xxvii) the fatty acid composition of the lipid is based on an efficiency of conversion of SDA to ETA acid by Δ6-elongase of at least about 60%, at least about 70%, at least about 75%, between about 60% and about 95%, between about 70% and about 88%, or between about 75% and about 85%,

[0044] xxviii) the fatty acid composition of the lipid is based on an efficiency of conversion of ETA to EPA by Δ5-desaturase of at least about 60%, at least about 70%, at least about 75%, between about 60% and about 99%, between about 70% and about 99%, or between about 75% and about 98%,

[0045] xxix) the fatty acid composition of the lipid is based on an efficiency of conversion of EPA to DPA by Δ5-elongase of at least about 80%, at least about 85%, at least about 90%, between about 50% and about 95%, or between about 85% and about 95%,

[0046] xxx) the fatty acid composition of the lipid is based on an efficiency of conversion of DPA to DHA by Δ4-desaturase of at least about 80%, at least about 90%, at least about 93%, between about 50% and about 95%, between about 80% and about 95%, or between about 85% and about 95%,

[0047] xxxi) the fatty acid composition of the lipid is based on an efficiency of conversion of oleic acid to DHA of at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 10% and about 30%, or between about 10% and about 25%,

[0048] xxxii) the fatty acid composition of the lipid is based on an efficiency of conversion of LA to DHA of at least about 15%, at least about 20%, at least about 22%, at least about 25%, between about 15% and about 50%, between about 20% and about 40%, or between about 20% and about 30%,

[0049] xxxiii) the fatty acid composition of the lipid is based on an efficiency of conversion of ALA to DHA of at least about 17%, at least about 22%, at least about 24%, between about 17% and about 55%, between about 22% and about 35%, or between about 24% and about 35%,

[0050] xxxiv) the total fatty acid in the extracted lipid has less than 1% C20:1,

[0051] xxxv) the triacylglycerol (TAG) content of the lipid is at least about 70%, at least about 80%, at least about 90%, at least 95%, between about 70% and about 99%, or between about 90% and about 99%,

[0052] xxxvi) the lipid comprises diacylglycerol (DAG),

[0053] xxxvii) the lipid comprises less than about 10%, less than about 5%, less than about 1%, or between about 0.001% and about 5%, free (non-esterified) fatty acids and/or phospholipid, or is essentially free thereof,

[0054] xxxviii) at least 70%, or at least 80%, of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG,

[0055] xxxix) the most abundant DHA-containing TAG species in the lipid is DHA/18:3/18:3 (TAG 58:12), and

[0056] xl) the lipid comprises tri-DHA TAG (TAG 66:18).

[0057] In another embodiment, the extracted lipid is in the form of an oil, wherein at least about 90%, or least about 95%, at least about 98%, or between about 95% and about 98%, by weight of the oil is the lipid.

[0058] In a preferred embodiment, the lipid or oil, preferably a seedoil, has the following features: in the total fatty acid content of the lipid or oil, the level of DHA is between about 7% and 20%, the level of palmitic acid is between about 2% and about 16%, the level of myristic acid is less than about 6%, the level of oleic acid is between about 1% and about 30%, the level of LA is between about 4% and about 35%, ALA is present, GLA is present, the level of SDA is between about 0.05% and about 7%, the level of ETA is less than about 4%, the level of EPA is between about 0.05% and about 10%, the level of DPA is between about 0.05% and about 8%, the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%, the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0, preferably less than about 0.50, the fatty acid composition of the lipid is based on: an efficiency of conversion of oleic acid to LA by Δ12-desaturase of at least about 60%, an efficiency of conversion of SDA to ETA acid by Δ6-elongase of at least about 60%, an efficiency of conversion of EPA to DPA by Δ5-elongase of between about 50% and about 95%, an efficiency of conversion of DPA to DHA by Δ4-desaturase of between about 50% and about 95%, an efficiency of conversion of oleic acid to DHA of at least about 10%, and the triacylglycerol (TAG) content of the lipid is at least about 70%, and optionally the lipid is essentially free of cholesterol and/or the lipid comprises tri-DHA TAG (TAG 66:18).

[0059] In a more preferred embodiment, the lipid or oil, preferably a seedoil, has the following features: in the total fatty acid content of the lipid, the level of DHA is between about 7% and 20%, the level of palmitic acid is between about 2% and about 16%, the level of myristic acid is less than about 2%, the level of oleic acid is between about 1% and about 30%, the level of LA is between about 4% and about 35%, the level of ALA is between about 7% and about 40%, the level of GLA is less than about 4%, the level of SDA is between about 0.05% and about 7%, the level of ETA is less than about 4%, the level of ETrA is between about 0.05% and about 4%, the level of EPA is between about 0.05% and about 10%, the level of DPA is between about 0.05% and about 8%, the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%, the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 0.5% and about 10%, the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between 36% and about 75%, the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 9% and about 33%, the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0, the fatty acid composition of the lipid is based on: an efficiency of conversion of oleic acid to LA by Δ12-desaturase of at least about 60%, an efficiency of conversion of SDA to ETA acid by Δ6-elongase of at least about 60%, an efficiency of conversion of ETA to EPA by Δ5-desaturase of at least about 60%, an efficiency of conversion of EPA to DPA by Δ5-elongase of between about 50% and about 95%, an efficiency of conversion of DPA to DHA by Δ4-desaturase of between about 50% and about 95%, an efficiency of conversion of oleic acid to DHA of at least about 10%, an efficiency of conversion of LA to DHA of at least about 15%, an efficiency of conversion of ALA to DHA of at least about 17%, and the total fatty acid content in the extracted lipid has less than 1% C20:1, the triacylglycerol (TAG) content of the lipid is at least about 70%, the lipid is essentially free of cholesterol, and the lipid comprises tri-DHA TAG (TAG 66:18). Preferably, the lipid or oil is canola oil and/or has not been treated with a transesterification process after it was extracted from the plant or plant part. In a particular embodiment, the lipid or canola oil may subsequently be treated to convert the fatty acids in the oil to alkyl esters such as methyl or ethyl esters. Further treatment may be applied to enrich the lipid or oil for the DHA.

[0060] In an embodiment, the lipid or oil, preferably a seedoil, has the following features: in the total fatty acid content of the lipid, the level of DHA is between about 7% and 20%, the level of palmitic acid is between about 2% and about 16%, the level of myristic acid is less than about 2%, the level of oleic acid is between about 30% and about 60%, preferably between about 45% and about 60%, the level of LA is between about 4% and about 20%, the level of ALA is between about 2% and about 16%, the level of GLA is less than about 3%, the level of SDA is less than about 3%, the level of ETA is less than about 4%, the level of ETrA less than about 2%, the level of EPA is less than about 4%, the level of DPA is less than about 4%, the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%, the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 30% and about 60%, or between about 40% and about 60%, the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 0.5% and about 10%, the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 10% and about 20%, the level of new of 3 fatty acids in the total fatty acid content of the extracted lipid is between about 9% and about 20%, the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, preferably less than about 0.50, the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0, the triacylglycerol (TAG) content of the lipid is at least about 70%, the lipid is essentially free of cholesterol, and the lipid comprises tri-DHA TAG (TAG 66:18). Preferably, the lipid or oil is essentially free of SDA, EPA and ETA and/or is canola oil and/or has not been treated with a transesterification process after it was extracted from the plant or plant part. In a particular embodiment, the lipid or canola oil may subsequently be treated to convert the fatty acids in the oil to alkyl esters such as methyl or ethyl esters. Further treatment may be applied to enrich the lipid or oil for the DHA.

[0061] In a further preferred embodiment, the lipid or oil, preferably a seedoil, has the following features: in the total fatty acid content of the lipid or oil, the level of DHA is between about 7% and 20%, the level of palmitic acid is between about 2% and about 16%, the level of myristic acid is less than about 6%, the level of oleic acid is between about 1% and about 30%, the level of LA is between about 4% and about 35%, ALA is present, GLA is present, the level of SDA is between about 0.05% and about 7%, the level of ETA is less than about 6%, the level of EPA is between about 0.05% and about 10%, the level of DPA is between about 0.05% and about 8%.

[0062] In a further embodiment, the extracted lipid further comprises one or more sterols, preferably plant sterols.

[0063] In another embodiment, the extracted lipid is in the form of an oil, and comprises less than about 10 mg of sterols/g of oil, less than about 7 mg of sterols/g of oil, between about 1.5 mg and about 10 mg of sterols/g of oil, or between about 1.5 mg and about 7 mg of sterols/g of oil.

[0064] Examples of sterols which can be in the extracted lipid include, but are not necessarily limited to, one or more or all of campesterol/24-methylcholesterol, Δ5-stigmasterol, eburicol, β-sitosterol/24-ethylcholesterol, Δ5-avenasterol/isofucosterol, Δ7-stigmasterol/stigmast-7-en-3β-ol, and Δ7-avenasterol.

[0065] In an embodiment, the plant species is one listed in Table 26, such as canola, and the level of sterols are about the same as that listed in Table 26 for that particular plant species.

[0066] In an embodiment, the extracted lipid comprises less than about 0.5 mg of cholesterol/g of oil, less than about 0.25 mg of cholesterol/g of oil, between about 0 mg and about 0.5 mg of cholesterol/g of oil, or between about 0 mg and about 0.25 mg of cholesterol/g of oil, or which is essentially free of cholesterol.

[0067] In a further embodiment, the lipid is an oil, preferably oil from an oilseed. Examples of such oils include, but are not limited to, Brassica sp. oil such as canola oil, Gossypium hirsutum oil, Linum usitatissimum oil, Helianthus sp. oil, Carthamus tinctorius oil, Glycine max oil, Zea mays oil, Arabidopsis thaliana oil, Sorghum bicolor oil, Sorghum vulgare oil, Avena sativa oil, Trifolium sp. oil, Elaesis guineenis oil, Nicotiana benthamiana oil, Hordeum vulgare oil, Lupinus angustifolius oil, Oryza sativa oil, Oryza glaberrima oil, Camelina sativa oil, Crambe abyssinica oil, Miscanthus x giganteus oil, or Miscanthus sinensis oil.

[0068] Also provided is extracted plant lipid, preferably extracted canola seedoil, comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein lipid has the following features in the total fatty acid content of the lipid;

[0069] i) the level of DHA is about 3%, about 4%, about 5%, about 6% or about 7%,

[0070] ii) the level of palmitic acid is between about 2% and about 16%,

[0071] iii) the level of myristic acid is less than about 2%,

[0072] iv) the level of oleic acid is between about 30% and about 60%, preferably between about 45% and about 60%,

[0073] v) the level of LA is between about 4% and about 20%,

[0074] vi) the level of ALA is between about 2% and about 16%,

[0075] vii) the level of GLA is less than about 4%,

[0076] viii) the level of SDA is less than about 6%, or less than about 4%,

[0077] ix) the level of ETA is less than about 6%, or less than about 4%,

[0078] x) the level of ETrA less than about 1%,

[0079] xi) the level of EPA is less than about 10% and/or the level of EPA is 0.5-2.0 fold the level of DHA,

[0080] xii) the level of DPA is less than about 4%,

[0081] xiii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%,

[0082] xiv) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 30% and about 70%,

[0083] xv) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 15% and about 75%, preferably between about 15% and about 30%,

[0084] xvi) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 0.5% and about 10%,

[0085] xvii) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 10% and about 20%,

[0086] xviii) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 3% and about 20%,

[0087] xix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, preferably less than about 0.50,

[0088] xx) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0,

[0089] xxi) the triacylglycerol (TAG) content of the lipid is at least about 70%, and

[0090] xxii) the lipid is essentially free of cholesterol. In an embodiment, the lipid comprises tri-DHA TAG (TAG 66:18). More preferably, the lipid is essentially free of SDA and ETA, and/or has not been treated with a transesterification process after it was extracted from the plant or plant part.

[0091] In another aspect, provided is extracted plant lipid, comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA) and docosahexaenoic acid (DHA), and one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein (i) the level of DHA in the total fatty acid content of the extracted lipid is between 7% and 20%, (ii) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 16%, (iii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 6%, (iv) the level of oleic acid in the total fatty acid content of the extracted lipid is between 1% and 30% or between 30% and 60%, (v) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between 4% and 35%, (vi) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between 4% and 40%, (vii) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than 4%, (viii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between 4% and 25%, (ix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between 1.0 and 3.0 or between 0.1 and 1, (x) the triacylglycerol (TAG) content of the lipid is at least 70%, and (xi) at least 70% of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG. In an embodiment, one or more or all of the following features

[0092] i) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 15%,

[0093] ii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 1%,

[0094] iii) the level of oleic acid in the total fatty acid content of the extracted lipid is between about 3% and about 30%, between about 6% and about 30%, between 1% and about 20%, between about 45% and about 60%, or is about 30%,

[0095] iv) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between about 4% and about 20%, or between about 4% and 17%,

[0096] v) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between about 7% and about 40%, between about 10% and about 35%, between about 20% and about 35%, or between about 4% and 16%,

[0097] vi) the level of γ-linolenic acid (GLA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, between 0.05% and 7%, between 0.05% and 4%, or between 0.05% and about 3%, or between 0.05% and about 2%,

[0098] vii) the level of stearidonic acid (SDA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, between about 0.05% and about 7%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between 0.05% and about 2%,

[0099] viii) the level of eicosatetraenoic acid (ETA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 1%, less than about 0.5%, between about 0.05% and about 5%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between about 0.05% and about 2%,

[0100] ix) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than about 2%, less than about 1%, between 0.05% and 4%, between 0.05% and 3%, or between 0.05% and about 2%, or between 0.05% and about 1%,

[0101] x) the level of eicosapentaenoic acid (EPA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, between 0.05% and 10%, between 0.05% and 5%, or between 0.05% and about 3%, or between 0.05% and about 2%,

[0102] xi) the level of docosapentaenoic acid (DPA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, between 0.05% and 8%, between 0.05% and 5%, or between 0.05% and about 3%. or between 0.05% and about 2%,

[0103] xii) the level of DHA in the total fatty acid content of the extracted lipid is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, between about 8% and 20%, between about 10% and 20%, between about 11% and 20%, between about 10% and about 16%, or between about 14% and

[0104] 20%,

[0105] xiii) the lipid comprises ω6-docosapentaenoic acid (22:5.sup.Δ4, 7, 10, 13, 16) in its fatty acid content,

[0106] xiv) the lipid is essentially free of ω6-docosapentaenoic acid (22:5.sup.Δ4, 7, 10, 13, 16) in its fatty acid content,

[0107] xv) the lipid is essentially free of SDA, EPA and ETA in its fatty acid content,

[0108] xvi) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 20%, or between about 6% and about 20%,

[0109] xvii) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, between about 8% and about 25%, or between 8% and about 22%,

[0110] xviii) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, between about 50% and about 75%, or between about 60% and about 75%,

[0111] xix) the level of total ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 35% and about 50%, between about 20% and about 35%, between about 6% and 20%, less than 20%, less than about 16%, less than about 10%, between about 1% and about 16%, between about 2% and about 10%, or between about 4% and about 10%,

[0112] xx) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is less than about 10%, less than about 8%, less than about 6%, less than 4%, between about 1% and about 20%, between about 1% and about 10%, between about 0.5% and about 8%, or between about 0.5% and 4%,

[0113] xxi) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between 36% and about 65%, between 40% and about 60%, between about 20% and about 35%, between about 10% and about 20%, about 25%, about 30%, about 35% or about 40%,

[0114] xxii) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between 9% and about 33%, between about 10% and about 20%, between about 20% and about 30%, between about 12% and about 25%, about 13%, about 15%, about 17% or about 20%,

[0115] xxiii) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 1.0, about 0.1 or about 0.2,

[0116] xxiv) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 1.0 and about 3.0, between about 0.1 and about 1, between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 0.1, about 0.2 or about 1.0,

[0117] xxv) the fatty acid composition of the lipid is based on an efficiency of conversion of oleic acid to DHA of at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 10% and about 30%, or between about 10% and about 25%,

[0118] xxvi) the fatty acid composition of the lipid is based on an efficiency of conversion of LA to DHA of at least about 15%, at least about 20%, at least about 22%, at least about 25%, between about 15% and about 50%, between about 20% and about 40%, or between about 20% and about 30%,

[0119] xxvii) the fatty acid composition of the lipid is based on an efficiency of conversion of ALA to DHA of at least about 17%, at least about 22%, at least about 24%, between about 17% and about 55%, between about 22% and about 35%, or between about 24% and about 35%,

[0120] xxviii) the total fatty acid in the extracted lipid has less than 1% C20:1,

[0121] xxix) the triacylglycerol (TAG) content of the lipid is at least about 80%, at least about 90%, at least 95%, between about 70% and about 99%, or between about 90% and about 99%,

[0122] xxx) the lipid comprises diacylglycerol (DAG),

[0123] xxxi) the lipid comprises less than about 10%, less than about 5%, less than about 1%, or between about 0.001% and about 5%, free (non-esterified) fatty acids and/or phospholipid, or is essentially free thereof,

[0124] xxxii) at least 80%, of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG,

[0125] xxxiii) the most abundant DHA-containing TAG species in the lipid is DHA/18:3/18:3 (TAG 58:12), and

[0126] xxxiv) the lipid comprises tri-DHA TAG (TAG 66:18).

[0127] With specific regard to the above aspect, in an embodiment

[0128] i) the lipid is in the form of an oil, wherein the oil comprises one or more sterols such as one or more or all of campesterol, Δ5-stigmasterol, eburicol, β-sitosterol, Δ5-avenasterol, Δ7-stigmasterol and Δ7-avenasterol, and optionally the oil comprises less than 10 mg of sterols/g of oil and/or the oil is essentially free of cholesterol, and/or

[0129] ii) the lipid is in the form of an oil from an oilseed such as oilseed is a Brassica sp oilseed or canola seed.

[0130] In another aspect, the present invention provides a process for producing extracted plant lipid, comprising the steps of

[0131] i) obtaining a plant part comprising lipid, the lipid comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of eicosapentaenoic acid (EPA), stearidonic acid (SDA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA in the total fatty acid content of extractable lipid in the plant part is about 7% to 20%, and

[0132] ii) extracting lipid from the plant part,

wherein the level of DHA in the total fatty acid content of the extracted lipid is about 7% to 20%.

[0133] In a preferred embodiment, the extracted lipid has one or more of the features defined above.

[0134] In an embodiment, wherein the plant part is a seed, preferably an oilseed. Examples of such seeds include, but are not limited to, Brassica sp., Gossypium hirsutum, Linum usitatissimum, Helianthus sp., Carthamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghum bicolor, Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifolium sp., Elaesis guineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinus angustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, or Crambe abyssinica, preferably a Brassica napus, B. juncea or C. sativa seed.

[0135] In another embodiment, the seed comprises at least about 18 mg, at least about 22 mg, at least about 26 mg, between about 18 mg and about 100 mg, between about 22 mg and about 70 mg, or between about 24 mg and about 50 mg, of DHA per gram of seed.

[0136] In a further embodiment, the plant part comprises exogenous polynucleotides encoding one of the following sets of enzymes;

[0137] i) an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,

[0138] ii) a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,

[0139] iii) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase,

[0140] iv) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase,

[0141] v) an ω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

[0142] vi) a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and a Δ5-elongase,

[0143] vii) a Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase, or

[0144] viii) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

and wherein each polynucleotide is operably linked to one or more promoters that are capable of directing expression of said polynucleotides in a cell of the plant part.

[0145] In yet a further embodiment, the plant part has one or more or all of the following features

[0146] i) the Δ12-desaturase converts oleic acid to linoleic acid in one or more cells of the plant with an efficiency of at least about 60%, at least about 70%, at least about 80%, between about 60% and about 98%, between about 70% and about 95%, or between about 75% and about 90%,

[0147] ii) the ω3-desaturase converts ω6 fatty acids to ω3 fatty acids in one or more cells of the plant with an efficiency of at least about 65%, at least about 75%, at least about 85%, between about 65% and about 95%, between about 75% and about 95%, or between about 80% and about 95%,

[0148] iii) the Δ6-desaturase converts ALA to SDA in one or more cells of the plant with an efficiency of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, between about 30% and about 70%, between about 35% and about 60%, or between about 50% and about 70%,

[0149] iv) the Δ6-desaturase converts linoleic acid to γ-linolenic acid in one or more cells of the plant with an efficiency of less than about 5%, less than about 2.5%, less than about 1%, between about 0.1% and about 5%, between about 0.5% and about 2.5%, or between about 0.5% and about 1%,

[0150] v) the Δ6-elongase converts SDA to ETA in one or more cells of the plant with an efficiency of at least about 60%, at least about 70%, at least about 75%, between about 60% and about 95%, between about 70% and about 88%, or between about 75% and about 85%,

[0151] vi) the Δ5-desaturase converts ETA to EPA in one or more cells of the plant with an efficiency of at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, between about 60% and about 99%, between about 70% and about 99%, or between about 75% and about 98%,

[0152] vii) the Δ5-elongase converts EPA to DPA in one or more cells of the plant with an efficiency of at least about 80%, at least about 85%, at least about 90%, between about 50% and about 95%, or between about 85% and about 95%,

[0153] viii) the Δ4-desaturase converts DPA to DHA in one or more cells of the plant with an efficiency of at least about 80%, at least about 90%, at least about 93%, between about 50% and about 95%, between about 80% and about 95%, or between about 85% and about 95%,

[0154] ix) the efficiency of conversion of oleic acid to DHA in one or more cells of the plant part is at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 10% and about 30%, or between about 10% and about 25%,

[0155] x) the efficiency of conversion of LA to DHA in one or more cells of the plant part is at least about 15%, at least about 20%, at least about 22%, at least about 25%, between about 15% and about 50%, between about 20% and about 40%, or between about 20% and about 30%,

[0156] xi) the efficiency of conversion of ALA to DHA in one or more cells of the plant part is at least about 17%, at least about 22%, at least about 24%, between about 17% and about 55%, between about 22% and about 35%, or between about 24% and about 35%,

[0157] xii) one or more cells of the plant part comprise at least about 15%, at least about 20%, between about 15% and about 30%, or between about 22.5% and about 27.5%, more ω3 fatty acids than corresponding cells lacking the exogenous polynucleotides,

[0158] xiii) the Δ6-desaturase preferentially desaturates α-linolenic acid (ALA) relative to linoleic acid (LA),

[0159] xiv) the Δ6-elongase also has Δ9-elongase activity,

[0160] xv) the Δ12-desaturase also has Δ15-desaturase activity,

[0161] xvi) the Δ6-desaturase also has Δ8-desaturase activity,

[0162] xvii) the Δ8-desaturase also has Δ6-desaturase activity or does not have Δ6-desaturase activity,

[0163] xviii) the Δ15-desaturase also has ω3-desaturase activity on GLA,

[0164] xix) the ω3-desaturase also has Δ15-desaturase activity on LA,

[0165] xx) the ω3-desaturase desaturates both LA and/or GLA,

[0166] xxi) the ω3-desaturase preferentially desaturates GLA relative to LA,

[0167] xxii) the level of DHA in the plant part is based on an efficiency of conversion of oleic acid to DHA in the plant part of at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 15% and about 30%, or between about 20% and about 25%,

[0168] xxiii) the level of DHA in the plant part is based on an efficiency of conversion of LA to DHA in the plant part of at least about 15%, at least about 20%, at least about 22%, between about 15% and about 60%, between about 20% and about 40%, or between about 22% and about 30%,

[0169] xxiv) the level of DHA in the plant part is based on an efficiency of conversion of ALA to DHA in the plant part of at least about 17%, at least about 22%, at least about 24%, between about 17% and about 65%, between about 22% and about 35%, or between about 24% and about 35%

[0170] xxx) one or more or all of the desaturases have greater activity on an acyl-CoA substrate than a corresponding acyl-PC substrate,

[0171] xxxi) the Δ6-desaturase has greater Δ6-desaturase activity on ALA than LA as fatty acid substrate,

[0172] xxxii) the Δ6-desaturase has greater Δ6-desaturase activity on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate,

[0173] xxxiii) the Δ6-desaturase has at least about a 2-fold greater Δ6-desaturase activity, at least 3-fold greater activity, at least 4-fold greater activity, or at least 5-fold greater activity, on ALA as a substrate compared to LA,

[0174] xxxiv) the Δ6-desaturase has greater activity on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate,

[0175] xxxv) the Δ6-desaturase has at least about a 5-fold greater Δ6-desaturase activity or at least 10-fold greater activity, on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate,

[0176] xxxvi) the desaturase is a front-end desaturase,

[0177] xxxvii) the Δ6-desaturase has no detectable Δ5-desaturase activity on ETA.

[0178] In yet a further embodiment, the plant part has one or more or all of the following features

[0179] i) the Δ12-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:10, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO: 10,

[0180] ii) the ω3-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:12, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:12,

[0181] iii) the Δ6-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:16, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:16,

[0182] iv) the Δ6-elongase comprises amino acids having a sequence as provided in SEQ ID NO:25, a biologically active fragment thereof such as SEQ ID NO:26, or an amino acid sequence which is at least 50% identical to SEQ ID NO:25 and/or SEQ ID NO:26,

[0183] v) the Δ5-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:30, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:30,

[0184] vi) the Δ5-elongase comprises amino acids having a sequence as provided in SEQ ID NO:37, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:37,

[0185] vii) the Δ4-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:41, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:41.

[0186] In an embodiment, the plant part further comprises an exogenous polynucleotide encoding a diacylglycerol acyltransferase (DGAT), monoacylglycerol acyltransferase (MGAT), glycerol-3-phosphate acyltransferase (GPAT), 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT) preferably an LPAAT which can use a C22 polyunsaturated fatty acyl-CoA substrate, acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), phospholipase A₂ (PLA₂), phospholipase C (PLC), phospholipase D (PLD), CDP-choline diacylglycerol choline phosphotransferase (CPT), phoshatidylcholine diacylglycerol acyltransferase (PDAT), phosphatidylcholine:diacylglycerol choline phosphotransferase (PDCT), acyl-CoA synthase (ACS), or a combination of two or more thereof.

[0187] In another embodiment, the plant part further comprises an introduced mutation or an exogenous polynucleotide which down regulates the production and/or activity of an endogenous enzyme in a cell of the plant part selected from FAE1, DGAT, MGAT, GPAT, LPAAT, LPCAT, PLA₂, PLC, PLD, CPT, PDAT, a thioesterase such as FATB, or a Δ12-desaturase, or a combination of two or more thereof.

[0188] In a further embodiment, at least one, or all, of the promoters are seed specific promoters. In an embodiment, at least one, or all, of the promoters have been obtained from oil biosynthesis or accumulation genes such as oleosin, or from seed storage protein genes such as conlinin.

[0189] In another embodiment, the promoter(s) directing expression of the exogenous polynucleotides encoding the Δ4-desaturase and the Δ5-elongase initiate expression of the polynucleotides in developing seed of the plant part before, or reach peak expression before, the promoter(s) directing expression of the exogenous polynucleotides encoding the Δ12-desaturase and the ω3-desaturase.

[0190] In a further embodiment, the exogenous polynucleotides are covalently linked in a DNA molecule, preferably a T-DNA molecule, integrated into the genome of cells of the plant part and preferably where the number of such DNA molecules integrated into the genome of the cells of the plant part is not more than one, two or three, or is two or three.

[0191] In yet another embodiment, the plant comprises at least two different, exogenous polynucleotides each encoding a Δ6-desaturase which have the same or different amino acid sequences.

[0192] In a further embodiment, the total oil content of the plant part comprising the exogenous polynucleotides is at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or between about 50% and about 80% of the total oil content of a corresponding plant part lacking the exogenous polynucleotides. In these embodiments, the maximum oil content may be about 100% of the oil content of a corresponding wild-type plant part.

[0193] In another embodiment, the lipid is in the form of an oil, preferably a seedoil from an oilseed, and wherein at least about 90%, or about least 95%, at least about 98%, or between about 95% and about 98%, by weight of the lipid is triacylglycerols.

[0194] In a further embodiment, the process further comprises treating the lipid to increase the level of DHA as a percentage of the total fatty acid content. For example, the treatment is transesterification. For example, the lipid such as canola oil may be treated to convert the fatty acids in the oil to alkyl esters such as methyl or ethyl esters, which may then be fractionated to enrich the lipid or oil for the DHA.

[0195] Further, provided is a process for producing extracted plant lipid, comprising the steps of

[0196] i) obtaining a plant part, preferably canola seed, comprising lipid, the lipid comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of eicosapentaenoic acid (EPA), stearidonic acid (SDA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA in the total fatty acid content of extractable lipid in the plant part is about 3%, about 4%, about 5%, about 6% or about 7%, and

[0197] ii) extracting lipid from the plant part,

wherein the extracted lipid has the following features in the total fatty acid content of the lipid;

[0198] i) the level of DHA is about 3%, about 4%, about 5%, about 6% or about 7%,

[0199] ii) the level of palmitic acid is between about 2% and about 16%,

[0200] iii) the level of myristic acid is less than about 2%,

[0201] iv) the level of oleic acid is between about 30% and about 60%, preferably between about 45% and about 60%,

[0202] v) the level of LA is between about 4% and about 20%,

[0203] vi) the level of ALA is between about 2% and about 16%,

[0204] vii) the level of GLA is less than about 4%,

[0205] viii) the level of SDA is less than about 6%, or less than about 4%,

[0206] ix) the level of ETA is less than about 6%, or less than about 4%,

[0207] x) the level of ETrA less than about 1%,

[0208] xi) the level of EPA is less than about 10% and/or the level of EPA is 0.5-2.0 fold the level of DHA,

[0209] xii) the level of DPA is less than about 4%,

[0210] xiii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 25%,

[0211] xiv) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 30% and about 70%,

[0212] xv) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 15% and about 75%, preferably between about 15% and about 30%,

[0213] xvi) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 0.5% and about 10%,

[0214] xvii) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 10% and about 20%,

[0215] xviii) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between about 3% and about 20%,

[0216] xix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.05 and about 3.0, preferably less than about 0.50,

[0217] xx) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.03 and about 3.0,

[0218] xxi) the triacylglycerol (TAG) content of the lipid is at least about 70%, and

[0219] xxii) the lipid is essentially free of cholesterol. In an embodiment, the lipid comprises tri-DHA TAG (TAG 66:18). More preferably, the lipid is essentially free of SDA and ETA, and/or has not been treated with a transesterification process after it was extracted from the plant or plant part.

[0220] Also provided is a process for producing extracted plant lipid, comprising the steps of

[0221] i) obtaining a plant part comprising lipid, the lipid comprising fatty acids in an esterified form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA) and docosahexaenoic acid (DHA), and one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein (i) the level of DHA in the total fatty acid content of the extracted lipid is between 7% and 20%, (ii) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 16%, (iii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 6%, (iv) the level of oleic acid in the total fatty acid content of the extracted lipid is between 1% and 30% or between 30% and 60%, (v) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between 4% and 35%, (vi) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between 4% and 40%, (vii) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than 4%, (viii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between 4% and 25%, (ix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between 1.0 and 3.0 or between 0.1 and 1, (x) the triacylglycerol (TAG) content of the lipid is at least 70%, and (xi) at least 70% of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG.

%, and

[0222] ii) extracting lipid from the plant part,

wherein the level of DHA in the total fatty acid content of the extracted lipid is about 7% to 20%.

[0223] Also provided is lipid, or oil comprising the lipid, produced using a process of the invention.

[0224] In another aspect, the present invention provides a process for producing ethyl esters of polyunsaturated fatty acids, the process comprising transesterifying triacylglycerols in extracted plant lipid, wherein the extracted plant lipid comprises fatty acids esterified in the form, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA), and docosahexaenoic acid (DHA), and optionally one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein the level of DHA in the total fatty acid content of the extracted lipid is about 7% to 20%, thereby producing the ethyl esters.

[0225] In a preferred embodiment, the extracted lipid has one or more of the features defined above.

[0226] In another aspect, the present invention provides a process for producing ethyl esters of polyunsaturated fatty acids, the process comprising transesterifying triacylglycerols in extracted plant lipid, wherein the extracted plant lipid comprises fatty acids esterified in the form of the triacylglycerols, the fatty acids comprising oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA), ω3 fatty acids which comprise α-linolenic acid (ALA) and docosahexaenoic acid (DHA), and one or more of stearidonic acid (SDA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and eicosatetraenoic acid (ETA), wherein (i) the level of DHA in the total fatty acid content of the extracted lipid is about 3%, about 4%, about 5%, about 6% or between 7% and 20%, (ii) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 16%, (iii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 6%, (iv) the level of oleic acid in the total fatty acid content of the extracted lipid is between 1% and 30% or between 30% and 60%, (v) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between 4% and 35%, (vi) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between 4% and 40%, (vii) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than 4%, (viii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between 4% and 25%, (ix) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between 1.0 and 3.0 or between 0.1 and 1, (x) the triacylglycerol (TAG) content of the lipid is at least 70%, and (xi) at least 70% of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG, thereby producing the ethyl esters. In an embodiment, the extracted plant lipid has one or more or all of the following features

[0227] i) the level of palmitic acid in the total fatty acid content of the extracted lipid is between 2% and 15%,

[0228] ii) the level of myristic acid (C14:0) in the total fatty acid content of the extracted lipid is less than 1%,

[0229] xxxv) the level of oleic acid in the total fatty acid content of the extracted lipid is between about 3% and about 30%, between about 6% and about 30%, between 1% and about 20%, between about 45% and about 60%, or is about 30%,

[0230] xxxvi) the level of linoleic acid (LA) in the total fatty acid content of the extracted lipid is between about 4% and about 20%, or between about 4% and 17%,

[0231] xxxvii) the level of α-linolenic acid (ALA) in the total fatty acid content of the extracted lipid is between about 7% and about 40%, between about 10% and about 35%, between about 20% and about 35%, or between about 4% and 16%,

[0232] xxxviii) the level of γ-linolenic acid (GLA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, between 0.05% and 7%, between 0.05% and 4%, or between 0.05% and about 3%, or between 0.05% and about 2%,

[0233] xxxix) the level of stearidonic acid (SDA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 3%, between about 0.05% and about 7%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between 0.05% and about 2%,

[0234] xl) the level of eicosatetraenoic acid (ETA) in the total fatty acid content of the extracted lipid is less than about 4%, less than about 1%, less than about 0.5%, between about 0.05% and about 5%, between about 0.05% and about 4%, between about 0.05% and about 3%, or between about 0.05% and about 2%,

[0235] xli) the level of eicosatrienoic acid (ETrA) in the total fatty acid content of the extracted lipid is less than about 2%, less than about 1%, between 0.05% and 4%, between 0.05% and 3%, or between 0.05% and about 2%, or between 0.05% and about 1%,

[0236] xlii) the level of eicosapentaenoic acid (EPA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, between 0.05% and 10%, between 0.05% and 5%, or between 0.05% and about 3%, or between 0.05% and about 2%,

[0237] xliii) the level of docosapentaenoic acid (DPA) in the total fatty acid content of the extracted lipid is less than 4%, less than about 3%, less than about 2%, between 0.05% and 8%, between 0.05% and 5%, or between 0.05% and about 3%, or between 0.05% and about 2%,

[0238] xliv) the level of DHA in the total fatty acid content of the extracted lipid is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, between about 8% and 20%, between about 10% and 20%, between about 11% and 20%, between about 10% and about 16%, or between about 14% and 20%.

[0239] xlv) the lipid comprises ω6-docosapentaenoic acid (22:5.sup.Δ4, 7, 10, 13, 16) in its fatty acid content,

[0240] xlvi) the lipid is essentially free of ω6-docosapentaenoic acid (22:5.sup.Δ4, 7, 10, 13, 16) in its fatty acid content,

[0241] xlvii) the lipid is essentially free of SDA, EPA and ETA in its fatty acid content,

[0242] xlviii) the level of total saturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 20%, or between about 6% and about 20%,

[0243] xlix) the level of total monounsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 4% and about 35%, between about 8% and about 25%, or between 8% and about 22%,

[0244] 1) the level of total polyunsaturated fatty acids in the total fatty acid content of the extracted lipid is between about 20% and about 75%, between about 50% and about 75%, or between about 60% and about 75%,

[0245] 1i) the level of total ω6 fatty acids in the total fatty acid content of the extracted lipid is between about 35% and about 50%, between about 20% and about 35%, between about 6% and 20%, less than 20%, less than about 16%, less than about 10%, between about 1% and about 16%, between about 2% and about 10%, or between about 4% and about 10%,

[0246] 1ii) the level of new ω6 fatty acids in the total fatty acid content of the extracted lipid is less than about 10%, less than about 8%, less than about 6%, less than 4%, between about 1% and about 20%, between about 1% and about 10%, between about 0.5% and about 8%, or between about 0.5% and 4%,

[0247] 1iii) the level of total ω3 fatty acids in the total fatty acid content of the extracted lipid is between 36% and about 65%, between 40% and about 60%, between about 20% and about 35%, between about 10% and about 20%, about 25%, about 30%, about 35% or about 40%,

[0248] 1iv) the level of new ω3 fatty acids in the total fatty acid content of the extracted lipid is between 9% and about 33%, between about 10% and about 20%, between about 20% and about 30%, between about 12% and about 25%, about 13%, about 15%, about 17% or about 20%,

[0249] 1v) the ratio of total ω6 fatty acids: total ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 1.0, about 0.1 or about 0.2,

[0250] 1vi) the ratio of new ω6 fatty acids: new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 1.0 and about 3.0, between about 0.1 and about 1, between about 0.1 and about 0.5, less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.15, about 0.1, about 0.2 or about 1.0,

[0251] 1vii) the fatty acid composition of the lipid is based on an efficiency of conversion of oleic acid to DHA of at least about 10%, at least about 15%, at least about 20%, between about 10% and about 50%, between about 10% and about 30%, or between about 10% and about 25%,

[0252] 1viii) the fatty acid composition of the lipid is based on an efficiency of conversion of LA to DHA of at least about 15%, at least about 20%, at least about 22%, at least about 25%, between about 15% and about 50%, between about 20% and about 40%, or between about 20% and about 30%,

[0253] 1ix) the fatty acid composition of the lipid is based on an efficiency of conversion of ALA to DHA of at least about 17%, at least about 22%, at least about 24%, between about 17% and about 55%, between about 22% and about 35%, or between about 24% and about 35%,

[0254] 1x) the total fatty acid in the extracted lipid has less than 1% C20:1,

[0255] 1xi) the triacylglycerol (TAG) content of the lipid is at least about 80%, at least about 90%, at least 95%, between about 70% and about 99%, or between about 90% and about 99%,

[0256] 1xii) the lipid comprises diacylglycerol (DAG),

[0257] 1xiii) the lipid comprises less than about 10%, less than about 5%, less than about 1%, or between about 0.001% and about 5%, free (non-esterified) fatty acids and/or phospholipid, or is essentially free thereof,

[0258] 1xiv) at least 80%, of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG,

[0259] 1xv) the most abundant DHA-containing TAG species in the lipid is DHA/18:3/18:3 (TAG 58:12), and

[0260] 1xvi) the lipid comprises tri-DHA TAG (TAG 66:18).

[0261] With specific regard to the above aspect, in an embodiment one or more or all of the following apply

[0262] i) the lipid is in the form of an oil, wherein the oil comprises one or more sterols such as one or more or all of campesterol, Δ5-stigmasterol, eburicol, β-sitosterol, Δ5-avenasterol, Δ7-stigmasterol and Δ7-avenasterol, and optionally the oil comprises less than 10 mg of sterols/g of oil and/or the oil is essentially free of cholesterol,

[0263] ii) the lipid is in the form of an oil from an oilseed such as oilseed is a Brassica sp oilseed or canola seed,

[0264] iii) the level of DHA in the total fatty acid content of the extracted plant lipid is about 3%, about 4%, about 5%, about 6%, or is between 7% and 20%.

[0265] In a further aspect, the present invention provides a chimeric genetic construct comprising in order a first gene, a second gene, a third gene, a fourth gene, a fifth gene and a sixth gene which are all covalently linked on a single DNA molecule,

wherein the first, second and third genes are joined together as a first gene cluster and the fourth, fifth and sixth genes are joined together as a second gene cluster, wherein each gene comprises a promoter, a coding region and a transcription terminator and/or polyadenylation region such that each promoter is operably linked to the coding region and transcription terminator and/or polyadenylation region, wherein each promoter is independently identical or different to the other promoters such that the DNA molecule comprises three, four, five or six different promoters, wherein one or more or all of the promoters are heterologous with respect to the coding region to which it is operably linked, wherein the direction of transcription of the first gene is away from the third gene and opposite to the direction of transcription of the third gene, wherein the direction of transcription of the fourth gene is away from the sixth gene and opposite to the direction of transcription of the sixth gene, wherein the direction of transcription of the second gene is the same as for the first gene or the third gene, wherein the direction of transcription of the fifth gene is the same as for the fourth gene or the sixth gene, wherein the transcription terminator and/or polyadenylation region of the second gene is spaced apart from the promoter of the first or third genes, whichever is closer, by a first spacer region of between about 0.2 and about 3.0 kilobases, wherein the first gene cluster is spaced apart from the second gene cluster by a second spacer region of between about 1.0 and about 10.0 kilobases, and wherein the transcription terminator and/or polyadenylation region of the fifth gene is spaced apart from the promoter of the fourth or sixth genes, whichever is closer, by a third spacer region of between about 0.2 and about 3.0 kilobases.

[0266] In an embodiment, the DNA molecule comprises a seventh gene which is spaced apart from the first gene cluster or the second gene cluster, whichever is closer, by a spacer region of between about 1.0 and about 10.0 kilobases.

[0267] In another embodiment, the DNA molecule comprises two or more different transcription terminator and/or polyadenylation regions.

[0268] In yet a further embodiment, at least one of the spacer regions comprises a matrix attachment region (MAR).

[0269] In a further embodiment, the DNA molecule comprises right and left border regions flanking the genes and is a T-DNA molecule.

[0270] In another embodiment, the genetic construct is in an Agrobacterium cell or is integrated into the genome of a plant cell.

[0271] In a preferred embodiment, at least one of the genes encodes a fatty acid desaturase or a fatty acid elongase.

[0272] In another embodiment, the genetic construct comprises genes encoding a set of enzymes as defined herein, and/or wherein one or more of the genes encode an enzyme as defined herein.

[0273] In a further aspect, the present invention provides an isolated and/or exogenous polynucleotide comprising:

[0274] i) a sequence of nucleotides selected from any one of SEQ ID NOs: 1 to 9, 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45, and/or

[0275] ii) a sequence of nucleotides which are at least 95% identical or 99% identical to one or more of the sequences set forth in SEQ ID NOs: 1 to 9, 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45.

[0276] In a particularly preferred embodiment, the isolated and/or exogenous polynucleotide comprises:

[0277] i) a sequence of nucleotides of SEQ ID NO: 2, and/or

[0278] ii) a sequence of nucleotides which are at least 95% identical or 99% identical to the sequence set forth in SEQ ID NO: 2.

[0279] In another aspect, the present invention provides a vector or genetic construct comprising the polynucleotide of the invention and/or the genetic construct of the invention.

[0280] In an embodiment, the sequence of nucleotides selected from any one of SEQ ID NOs: 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45, or the sequence of nucleotides which is at least 95% identical or 99% identical to one or more of the sequences set forth in SEQ ID NOs: 11, 14, 18, 22, 23, 28, 34, 35, 39 or 45, is operably linked to a promoter.

[0281] In a further aspect, the present invention provides a host cell comprising exogenous polynucleotides encoding one of the following sets of enzymes;

[0282] i) an ω3-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,

[0283] ii) a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and a Δ5-elongase,

[0284] iii) a Δ12-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase,

[0285] iv) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase,

[0286] v) an ω3-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

[0287] vi) a Δ5-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and a Δ5-elongase,

[0288] vii) a Δ12-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase, or

[0289] viii) a Δ12-desaturase, a ω3-desaturase or a Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

and wherein each polynucleotide is operably linked to one or more promoters that are capable of directing expression of said polynucleotides in the cell.

[0290] In an embodiment, the cell comprises lipid as defined above, or wherein one or more or all of the desaturases or elongases have one or more of the features as defined above.

[0291] In another aspect, the present invention provides a host cell comprising

[0292] i) a first exogenous polynucleotide encoding a Δ12-desaturase which comprises amino acids having a sequence as provided in SEQ ID NO:10, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:10, and

[0293] ii) a second exogenous polynucleotide encoding a ω3-desaturase which comprises amino acids having a sequence as provided in SEQ ID NO: 12, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:12,

wherein each polynucleotide is operably linked to one or more promoters that are capable of directing expression of said polynucleotides in the cell.

[0294] In a further aspect, the present invention provides a host cell comprising one or more of the polynucleotide of the invention, the genetic construct of the invention, or the vector or genetic construct of the invention.

[0295] In an embodiment, the cell is in a plant, in a plant part and/or is a mature plant seed cell.

[0296] In an embodiment, the plant or plant seed is an oilseed plant or an oilseed, respectively.

[0297] Also provided is a transgenic non-human organism comprising a cell of the invention. Preferably, the transgenic non-human organism is a transgenic plant, preferably an oilseed plant or Arabidopsis thaliana. In an embodiment, the plant is a Brassica plant, preferably B. napus or B. juncea, or a plant other than Arabidopsis thaliana.

[0298] In another aspect, the present invention provides an oilseed plant comprising

[0299] a) lipid in its seed, the lipid comprising fatty acids in an esterified form, and

[0300] b) exogenous polynucleotides encoding one of the following sets of enzymes;

[0301] i) a Δ12-desaturase, a fungal ω3-desaturase and/or fungal Δ15-desaturase, a Δ6-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ6-elongase and an Δ5-elongase, or

[0302] ii) a Δ12-desaturase, a fungal o 3-desaturase and/or fungal Δ15-desaturase, a Δ8-desaturase, a Δ5-desaturase, a Δ4-desaturase, a Δ9-elongase and an Δ5-elongase,

[0303] wherein each polynucleotide is operably linked to one or more seed-specific promoters that are capable of directing expression of said polynucleotides in developing seed of the plant, wherein the fatty acids comprise oleic acid, palmitic acid, ω6 fatty acids which comprise linoleic acid (LA) and γ-linolenic acid (GLA), ω3 fatty acids which comprise α-linolenic acid (ALA), stearidonic acid (SDA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), and optionally eicosapentaenoic acid (EPA) and/or eicosatetraenoic acid (ETA), and wherein the level of DHA in the total fatty acid content of the lipid is about 7% to 20%.

[0304] Examples of oilseed plants include, but are not limited to, Brassica sp., Gossypium hirsutum, Linum usitatissimum, Helianthus sp., Carthamus tinctorius, Glycine max, Zea mays, Arabidopsis thaliana, Sorghum bicolor, Sorghum vulgare, Avena sativa, Trifolium sp., Elaesis guineenis, Nicotiana benthamiana, Hordeum vulgare, Lupinus angustifolius, Oryza sativa, Oryza glaberrima, Camelina sativa, or Crambe abyssinica. In an embodiment, the oilseed plant is a canola, Glycine max, Camelina sativa or Arabidopsis thaliana plant. In an alternate embodiment, the oilseed plant is other than A. thaliana.

[0305] In an embodiment, one or more of the desaturases is capable of using an acyl-CoA substrate. In a preferred embodiment, one or more of the Δ6-desaturase, Δ5-desaturase, Δ4-desaturase and Δ8-desaturase, if present, is capable of using an acyl-CoA substrate, preferably each of the i) Δ6-desaturase, Δ5-desaturase and Δ4-desaturase or ii) Δ5-desaturase, Δ4-desaturase and Δ8-desaturase is capable of using an acyl-CoA substrate. In an embodiment, a Δ12-desaturase and/or an ω3-desaturase is capable of using an acyl-CoA substrate. The acyl-CoA substrate is preferably an ALA-CoA, ETA-CoA, DPA-CoA, ETrA-CoA, LA-CoA, GLA-CoA, or ARA-CoA.

[0306] In an embodiment, mature, harvested seed of the plant has a DHA content of at least about 28 mg per gram seed, preferably at least about 32 mg per gram seed, at least about 36 mg per gram seed, at least about 40 mg per gram seed, more preferably at least about 44 mg per gram seed or at least about 48 mg per gram seed. The maximum DHA content may be about 80 to about 100 mg per gram seed, or about 80 mg or about 100 mg per gram seed.

[0307] In a further aspect, the present invention provides a Brassica napus, B. juncea or Camelina sativa plant which is capable of producing seed comprising DHA, wherein mature, harvested seed of the plant has a DHA content of at least about 28 mg per gram seed, preferably at least about 32 mg per gram seed, at least about 36 mg per gram seed, at least about 40 mg per gram seed, more preferably at least about 44 mg per grain seed or at least about 48 mg per gram seed. The maximum DHA content may be about 80 to about 100 mg per gram seed, or about 80 mg or about 100 mg per gram seed.

[0308] In another aspect, the present invention provides plant cell of a plant of the invention comprising the exogenous polynucleotides.

[0309] Also provided is a plant part, preferably a seed, which has one or more of the following features

[0310] i) is from a plant of the invention,

[0311] ii) comprises lipid as defined herein,

[0312] iii) can be used in a process of the invention,

[0313] iv) comprises a genetic construct of the invention, or

[0314] v) comprises a set of exogenous polynucleotides as defined herein.

[0315] In yet another aspect, the present invention provides mature, harvested Brassica napus, B. juncea or Camelina sativa seed comprising DHA and a moisture content of between about 4% and about 15% by weight, wherein the DHA content of the seed at least about 28 mg per gram seed, preferably at least about 32 mg per gram seed, at least about 36 mg per gram seed, at least about 40 mg per gram seed, more preferably at least about 44 mg per gram seed or at least about 48 mg per gram seed. The maximum DHA content may be about 80 to about 100 mg per gram seed, or about 80 mg or about 100 mg per gram seed.

[0316] In an embodiment, the cell of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, or the seed of the invention, which can be used to produce extracted lipid comprising one or more or all of the features defined herein.

[0317] In yet a further aspect, the present invention provides a method of producing a cell of the invention, the method comprising

[0318] a) introducing into the cell, preferably a cell which is not capable of synthesising a LC-PUFA, the gene construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, one or more of the combinations of exogenous polynucleotides defined herein,

[0319] b) optionally, expressing the genes or polynucleotide(s) in the cell;

[0320] c) optionally, analysing the fatty acid composition of the cell, and

[0321] d) optionally, selecting a cell which express the genes or polynucleotide(s).

[0322] In an embodiment, the lipid in the cell has one or more of the features defined herein.

[0323] In another embodiment, the gene construct, the isolated and/or exogenous polynucleotide, the vector, the genetic construct or combinations of exogenous polynucleotides, become stably integrated into the genome of the cell.

[0324] In a further embodiment, the cell is a plant cell, and the method further comprises the step of regenerating a transformed plant from the cell of step a).

[0325] In another embodiment, the genes and/or exogenous polynucleotide(s) are expressed transiently in the cell.

[0326] Also provided is a cell produced using a method of the invention.

[0327] In another aspect, the present invention provides a method of producing seed, the method comprising,

[0328] a) growing a plant of the invention, or a plant which produces a part as defined herein, preferably in a field as part of a population of at least 1000 such plants or in an area of at least 1 hectare planted at a standard planting density,

[0329] b) harvesting seed from the plant or plants, and

[0330] c) optionally, extracting lipid from the seed, preferably to produce oil with a total DHA yield of at least 60 kg DHA/hectare.

[0331] In an embodiment, the plant, plant cell, plant part or seed of the invention has one or more of the following features

[0332] i) the oil is as defined herein,

[0333] ii) the plant part or seed is capable of being used in a process of the invention,

[0334] iii) the exogenous polynucleotides are comprised in a genetic construct of the invention,

[0335] iv) the exogenous polynucleotides comprise an exogenous polynucleotide of the invention,

[0336] v) the plant cell is a cell of the invention, and

[0337] vi) the seed was produced according to the method of the invention.

[0338] In another aspect, the present invention provides a method of producing one or more fatty acid desaturases and/or fatty acid elongases, or one or more fatty acid desaturases and one or more fatty acid elongases, the method comprising expressing in a cell or cell free expression system the gene construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, one or more of the combinations of exogenous polynucleotides defined herein, preferably in a developing oilseed in an oilseed plant in the field.

[0339] In a further aspect, the present invention provides lipid, or oil, produced by, or obtained from, using the process of the invention, the cell of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, or the plant, plant cell, plant part or seed of the invention.

[0340] In an embodiment, the lipid or oil is obtained by extraction of oil from an oilseed. Examples of oil from oilseeds include, but are not limited to, canola oil (Brassica napus, Brassica rapa ssp.), mustard oil (Brassica juncea), other Brassica oil, sunflower oil (Helianthus annus), linseed oil (Linum usitatissimum), soybean oil (Glycine max), safflower oil (Carthamus tinctorius), corn oil (Zea mays), tobacco oil (Nicotiana tabacum), peanut oil (Arachis hypogaea), palm oil, cottonseed oil (Gossypium hirsutum), coconut oil (Cocos nucifera), avocado oil (Persea americana), olive oil (Olea europaea), cashew oil (Anacardium occidentale), macadamia oil (Macadamia intergrifolia), almond oil (Prunus amygdalus) or Arabidopsis seed oil (Arabidopsis thaliana).

[0341] In a further aspect, the present invention provides fatty acid produced by, or obtained from, using the process of the invention, the cell of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, or the plant, plant cell, plant part or seed of the invention. Preferably the fatty acid is DHA. The fatty acid may be in a mixture of fatty acids having a fatty acid composition as described herein. In an embodiment, the fatty acid is non-esterified.

[0342] Also provided is seedmeal obtained from seed of the invention. Preferred seedmeal includes, but not necessarily limited to, Brassica napus, B. juncea, Camelina sativa or Glycine max seedmeal. In an embodiment, the seedmeal comprises an exogenous polynucleotide(s) and/or genetic constructs as defined herein.

[0343] In another aspect, the present invention provides a composition comprising one or more of a lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, or the seedmeal of the invention. In embodiments, the composition comprises a carrier suitable for pharmaceutical, food or agricultural use, a seed treatment compound, a fertiliser, another food or feed ingredient, or added protein or vitamins.

[0344] Also provided is feedstuffs, cosmetics or chemicals comprising one or more of the lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, the seedmeal of the invention, or the composition of the invention.

[0345] In another aspect, the present invention provides a method of producing a feedstuff, the method comprising mixing one or more of the lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, the seedmeal of the invention, or the composition of the invention, with at least one other food ingredient.

[0346] In another aspect, the present invention provides a method of treating or preventing a condition which would benefit from a PUFA, the method comprising administering to a subject one or more of the lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, the seedmeal of the invention, the composition of the invention, or the feedstuff of the invention.

[0347] Examples of conditions which would benefit from a PUFA include, but are not limited to, cardiac arrhythmia's, angioplasty, inflammation, asthma, psoriasis, osteoporosis, kidney stones, AIDS, multiple sclerosis, rheumatoid arthritis, Crohn's disease, schizophrenia, cancer, foetal alcohol syndrome, attention deficient hyperactivity disorder, cystic fibrosis, phenylketonuria, unipolar depression, aggressive hostility, adrenoleukodystophy, coronary heart disease, hypertension, diabetes, obesity, Alzheimer's disease, chronic obstructive pulmonary disease, ulcerative colitis, restenosis after angioplasty, eczema, high blood pressure, platelet aggregation, gastrointestinal bleeding, endometriosis, premenstrual syndrome, myalgic encephalomyelitis, chronic fatigue after viral infections or an ocular disease.

[0348] Also provided is the use of one or more of the lipid or oil of the invention, the fatty acid of the invention, the genetic construct of the invention, the isolated and/or exogenous polynucleotide of the invention, the vector or genetic construct of the invention, the cell according of the invention, the transgenic organism of the invention, the oilseed plant of the invention, the Brassica napus, B. juncea or Camelina sativa plant of the invention, the plant part of the invention, the seed of the invention, the plant, plant cell, plant part or seed of the invention, the seedmeal of the invention, the composition of the invention, or the feedstuff of the invention for the manufacture of a medicament for treating or preventing a condition which would benefit from a PUFA. The production of the medicament may comprise mixing the oil of the invention with a pharmaceutically acceptable carrier, for treatment of a condition as described herein. The method may comprise firstly purifying the oil and/or transesterification, and/or fractionation of the oil to increase the level of DHA. In a particular embodiment, the method comprises treating the lipid or oil such as canola oil to convert the fatty acids in the oil to alkyl esters such as methyl or ethyl esters. Further treatment such as fractionation or distillation may be applied to enrich the lipid or oil for the DHA. In a preferred embodiment, the medicament comprises ethyl esters of DHA. In an even more preferred embodiment, the level of ethyl esters of DHA in the medicament is between 30% and 50%. The medicament may further comprise ethyl esters of EPA, such as between 30% and 50% of the total fatty acid content in the medicament. Such medicaments are suitable for administration to human or animal subjects for treatment of medical conditions as described herein.

[0349] In another aspect, the present invention provides a method of trading seed, comprising obtaining seed of the invention, and trading the obtained seed for pecuniary gain.

[0350] In an embodiment, obtaining the seed comprises cultivating plants of the invention and/or harvesting the seed from the plants.

[0351] In another embodiment, obtaining the seed further comprises placing the seed in a container and/or storing the seed.

[0352] In a further embodiment, obtaining the seed further comprises transporting the seed to a different location.

[0353] In yet another embodiment, the method further comprises transporting the seed to a different location after the seed is traded.

[0354] In a further embodiment, the trading is conducted using electronic means such as a computer.

[0355] In yet a further aspect, the present invention provides a process of producing bins of seed comprising:

[0356] a) swathing, windrowing and/or or reaping above-ground parts of plants comprising seed of the invention,

[0357] b) threshing and/or winnowing the parts of the plants to separate the seed from the remainder of the plant parts, and

[0358] c) sifting and/or sorting the seed separated in step b), and loading the sifted and/or sorted seed into bins, thereby producing bins of seed.

[0359] In an embodiment, where relevant, the lipid or oil, preferably seedoil, of, or useful for, the invention has fatty levels about those provided in a Table in the Examples section, such as seed 14 of Table 16.

[0360] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0361] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

[0362] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0363] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0364] FIG. 1. Aerobic DHA biosynthesis pathways.

[0365] FIG. 2. Map of the T-DNA insertion region between the left and right borders of pJP3416-GA7. RB denotes right border; LB, left border; TER, transcription terminator/polyadenylation region; PRO, promoter; Coding regions are indicated above the arrows, promoters and terminators below the arrows. Micpu-Δ6D, Micromonas pusilla Δ6-desaturase; Pyrco-Δ6E, Pyramimonas cordata Δ6-elongase; Pavsa-Δ5D, Pavlova salina Δ5-desaturase; Picpa-ω3D, Pichia pastoris ω3-desaturase; Pavsa-Δ4D, P. salina Δ4-desaturase; Lackl-Δ12D, Lachancea kluyveri Δ12-desaturase; Pyrco-Δ5E, Pyramimonas cordata Δ5-elongase. NOS denotes the Agrobacterium tumefaciens nopaline synthase transcription terminator/polyadenylation region; FP1, Brassica napus truncated napin promoter; FAE1, Arabidopsis thaliana FAE1 promoter; Lectin, Glycine max lectin transcription terminator/polyadenylation region; Cnl1 and Cnl2 denotes the Linum usitatissimum conlinin1 or conlinin2 promoter or terminator. MAR denotes the Rb7 matrix attachment region from Nicotiana tabacum.

[0366] FIG. 3. Map of the T-DNA insertion region between the left and right borders of pJP3404. Labels are as in FIG. 2.

[0367] FIG. 4. Map of the insertion region between the left and right borders of pJP3367. Labels are as in FIG. 2.

[0368] FIG. 5. DHA levels as a percentage of total fatty acids in seed lipid from multiple independent transgenic Arabidopsis thaliana seeds in both the T₂ and T₃ generations. The bracketed T₂ events were taken to T₃. Events from both the Columbia and fad2 mutant A. thaliana backgrounds are shown.

[0369] FIG. 6. Oil content (w/w) vs. DHA content, as a percentage of total fatty acid content of lipid from transgenic Arabidopsis thaliana seeds.

[0370] FIG. 7. Representative RT-PCR gel showing the low expression of the Δ6-desaturase gene relative to the other transgenes in the T-DNA of B. napus embryos transformed using pJP3416-GA7. Lanes from the left show RT-PCR products: 1, DNA size markers; lane 2, Δ12 desaturase; lane 3, ω3-desaturase; lane 4, Δ6-desaturase (low expression); lane 5, Δ6-elongase; lane 6, Δ5-desaturase; lane 7, Δ5-elongase; lane 8, Δ4-desaturase.

[0371] FIG. 8. Percentage of ALA plotted against percentage of oleic acid, each as a percentage of total fatty acids in lipid obtained from transgenic 35S:LEC2 Brassica napus somatic embryos.

[0372] FIG. 9. Positional distribution analysis by NMR on A) Tuna oil and, B) transgenic DHA Arabidopsis seed oil. The peaks labelled `DHA-alpha` represent the amount of DHA present at the sn-1 and sn-3 positions of TAG (with no positional preference this would equal 66% of total DHA) whilst the peaks labelled `DHA-beta` represent the amount of DHA present at the sn-2 position of TAG (with no preference this would equal 33% of DHA).

[0373] FIG. 10. LC-MS analysis of major DHA-containing triacylglycerol species in transgenic A. thaliana developing (grey) and mature (black) seeds. The number following the DHA denotes the total number of carbon atoms and total number of double bonds in the other two fatty acids. Therefore DHA/34:1 can also be designated TAG 56:7, etc.

[0374] FIG. 11. Map of the T-DNA insertion region between the left and right borders of pORE04+11ABGBEC_Cowpea_EPA_insert. Labels are as in FIG. 2; SSU, Arabidopsis thaliana rubisco small subunit promoter.

[0375] FIG. 12. Map of the binary vector pJP3364 showing the NotI restriction site into which the candidate Δ12-desaturases were cloned.

[0376] FIG. 13. BoxPlot generated using SigmaPlot showing the percentage of fatty acid 20:4ω6 (ARA) in seed lipid of Arabidopsis T2 seed populations transformed with pFN045-pFN050. The boundary of each box closest to zero indicates the 25th percentile, a line within each box marks the median, and the boundary of each box farthest from zero indicates the 75th percentile. Error bars shown above and below each box indicate the 90th and 10th percentiles.

[0377] FIG. 14. Average level of ARA as a percentage of the total fatty acid content in seed lipid of Arabidopsis T2 seed transformed with pFN045-pFN050.

[0378] FIG. 15. BoxPlot showing the percentage of fatty acid 20:2ω6 (EDA) in seed lipid of Arabidopsis T2 seed populations transformed with pFN045-pFN050. The BoxPlot represents values as described in FIG. 13.

[0379] FIG. 16. BoxPlot showing the percentage of ARA in seed lipid of Arabidopsis T4 seed populations transformed with pFN045-pFN050. The BoxPlot represents values as described in FIG. 13.

[0380] FIG. 17. Average level of ARA as a percentage of the total fatty acid content in seed lipid of Arabidopsis T4 seed populations transformed with pFN045-pFN050.

[0381] FIG. 18. BoxPlot showing the percentage of EDA in seed lipid of Arabidopsis T4 seed populations transformed with pFN045-pFN050. The BoxPlot represents values as described in FIG. 13.

[0382] FIG. 19. (A) Basic phytosterol structure with ring and side chain numbering. (B) Chemical structures of some of the phytosterols.

[0383] FIG. 20. Phylogenetic tree of known LPAATs.

[0384] FIG. 21. The various acyl exchange enzymes which transfer fatty acids between PC, CoA pools, and TAG pools. Adapted from Singh et al. (2005),

KEY TO THE SEQUENCE LISTING

[0385] SEQ ID NO:1--pJP3416-GA7 nucleotide sequence.

[0386] SEQ ID NO:2--pGA7-mod_B nucleotide sequence.

[0387] SEQ ID NO:3--pGA7-mod_C nucleotide sequence.

[0388] SEQ ID NO:4--pGA7-mod_D nucleotide sequence.

[0389] SEQ ID NO:5--pGA7-mod_E nucleotide sequence.

[0390] SEQ ID NO:6--pGA7-mod_F nucleotide sequence.

[0391] SEQ ID NO:7--pGA7-mod_G nucleotide sequence.

[0392] SEQ ID NO:8--pORE04+1ABGBEC_Cowpea_EPA_insert nucleotide sequence.

[0393] SEQ ID NO:9--Codon-optimized open reading frame for expression of Lachancea kluyveri Δ12 desaturase in plants.

[0394] SEQ ID NO:10--Lachancea kluyveri Δ12-desaturase.

[0395] SEQ ID NO: 1--Codon-optimized open reading frame for expression of Pichia pastoris ω3 desaturase in plants.

[0396] SEQ ID NO:12--Pichia pastoris ω3 desaturase.

[0397] SEQ ID NO: 13--Open reading frame encoding Micromonas pusilla Δ6-desaturase.

[0398] SEQ ID NO:14--Codon-optimized open reading frame for expression of Micromonas pusilla Δ6-desaturase in plants (version 1).

[0399] SEQ ID NO: 15--Codon-optimized open reading frame for expression of Micromonas pusilla Δ6-desaturase in plants (version 2).

[0400] SEQ ID NO: 16--Micromonas pusilla Δ6-desaturase.

[0401] SEQ ID NO:17--Open reading frame encoding Ostreococcus lucimarinus Δ6-desaturase.

[0402] SEQ ID NO: 18--Codon-optimized open reading frame for expression of Ostreococcus lucimarinus Δ6-desaturase in plants.

[0403] SEQ ID NO: 19--Ostreococcus lucimarinus Δ6-desaturase.

[0404] SEQ ID NO:20--Ostreococcus tauri Δ6-desaturase.

[0405] SEQ ID NO:21--Open reading frame encoding Pyramimonas cordata Δ6-elongase.

[0406] SEQ ID NO:22--Codon-optimized open reading frame for expression of Pyramimonas cordata Δ6-elongase in plants (truncated at 3' end and encoding functional elongase) (version 1).

[0407] SEQ ID NO:23--Codon-optimized open reading frame for expression of Pyramimonas cordata Δ6-elongase in plants (truncated at 3' end and encoding functional elongase) (version 2).

[0408] SEQ ID NO:24--Codon-optimized open reading frame for expression of Pyramimonas cordata Δ6-elongase in plants (truncated at 3' end and encoding functional elongase) (version 3).

[0409] SEQ ID NO:25--Pyramimonas cordata Δ6-elongase.

[0410] SEQ ID NO:26--Truncated Pyramimonas cordata Δ6-elongase.

[0411] SEQ ID NO:27--Open reading frame encoding Pavlova salina Δ5-desaturase.

[0412] SEQ ID NO:28--Codon-optimized open reading frame for expression of Pavlova salina Δ5-desaturase in plants (version 1).

[0413] SEQ ID NO:29--Codon-optimized open reading frame for expression of Pavlova salina Δ5-desaturase in plants (version 2).

[0414] SEQ ID NO:30--Pavlova salina Δ5-desaturase.

[0415] SEQ ID NO:31--Open reading frame encoding Pyramimonas cordata Δ5-desaturase.

[0416] SEQ ID NO:32--Pyramimonas cordata Δ5-desaturase.

[0417] SEQ ID NO:33--Open reading frame encoding Pyramimonas cordata Δ5-elongase.

[0418] SEQ ID NO:34--Codon-optimized open reading frame for expression of Pyramimonas cordata Δ5-elongase in plants (version 1).

[0419] SEQ ID NO:35--Codon-optimized open reading frame for expression of Pyramimonas cordata Δ5-elongase in plants (version 2).

[0420] SEQ ID NO:36--Codon-optimized open reading frame for expression of Pyramimonas cordata Δ5-elongase in plants (version 3).

[0421] SEQ ID NO:37--Pyramimonas cordata Δ5-elongase.

[0422] SEQ ID NO:38--Open reading frame encoding Pavlova salina Δ4-desaturase.

[0423] SEQ ID NO:39--Codon-optimized open reading frame for expression of Pavlova salina Δ4-desaturase in plants (version 1).

[0424] SEQ ID NO:40--Codon-optimized open reading frame for expression of Pavlova salina Δ4-desaturase in plants (version 2).

[0425] SEQ ID NO:41--Pavlova salina Δ4-desaturase.

[0426] SEQ ID NO:42--Open reading frame encoding Isochrysis galbana Δ9-elongase.

[0427] SEQ ID NO:43--Isochrysis galbana Δ9-elongase.

[0428] SEQ ID NO:44--Open reading frame encoding Emiliania huxleyi CCMP1516 Δ9-elongase.

[0429] SEQ ID NO:45--Codon-optimized open reading frame for expression of Emiliania huxleyi Δ9-elongase in plants.

[0430] SEQ ID NO:46--Emiliania huxleyi CCMP1516 Δ9-elongase.

[0431] SEQ ID NO:47--Open reading frame encoding Pavlova pinguis Δ9-elongase.

[0432] SEQ ID NO:48--Pavlova pinguis Δ9-elongase.

[0433] SEQ ID NO:49--Open reading frame encoding Pavlova salina Δ9-elongase.

[0434] SEQ ID NO:50--Pavlova salina Δ9-elongase.

[0435] SEQ ID NO:51--Open reading frame encoding Pavlova salina Δ8-desaturase.

[0436] SEQ ID NO:52--Pavlova salina Δ8-desaturase.

[0437] SEQ ID NO:53--P19 viral suppressor.

[0438] SEQ ID NO:54--V2 viral suppressor.

[0439] SEQ ID NO:55--P38 viral suppressor.

[0440] SEQ ID NO:56--Pe-P0 viral suppressor.

[0441] SEQ ID NO:57--RPV-P0 viral suppressor.

[0442] SEQ ID NO:58--Open reading frame encoding P19 viral suppressor.

[0443] SEQ ID NO:59--Open reading frame encoding V2 viral suppressor.

[0444] SEQ ID NO:60--Open reading frame encoding P38 viral suppressor.

[0445] SEQ ID NO:61--Open reading frame encoding Pe-P0 viral suppressor.

[0446] SEQ ID NO:62--Open reading frame encoding RPV-P0 viral suppressor.

[0447] SEQ ID NO: 63--Arabidopsis thaliana LPAAT2.

[0448] SEQ ID NO: 64--Limnanthes alba LPAAT.

[0449] SEQ ID NO: 65--Saccharomyces cerevisiae LPAAT.

[0450] SEQ ID NO: 66--Micromonas pusilla LPAAT.

[0451] SEQ ID NO: 67--Mortierella alpina LPAAT.

[0452] SEQ ID NO: 68--Brassica napus LPAAT.

[0453] SEQ ID NO: 69--Brassica napus LPAAT.

[0454] SEQ ID NO: 70--Phytophthora infestans ω3 desaturase.

[0455] SEQ ID NO: 71--Thalassiosira pseudonana ω3 desaturase.

[0456] SEQ ID NO: 72--Pythium irregulare ω3 desaturase.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Definitions

[0457] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, fatty acid synthesis, transgenic plants, protein chemistry, and biochemistry).

[0458] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors), Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0459] The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

[0460] As used herein, the term "about", unless stated to the contrary, refers to +/-10%, more preferably +/-5%, more preferably +/-1% of the designated value.

[0461] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

SELECTED DEFINITIONS

[0462] As used herein, the terms "extracted plant lipid" and "isolated plant lipid" refer to a lipid composition which has been extracted from, for example by crushing, a plant or part thereof such as seed. The extracted lipid can be a relatively crude composition obtained by, for example, crushing a plant seed, or a more purified composition where most, if not all, of one or more or each of the water, nucleic acids, proteins and carbohydrates derived from the plant material have been removed. Examples of purification methods are described below. In an embodiment, the extracted or isolated plant lipid comprises at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% (w/w) lipid by weight of the composition. The lipid may be solid or liquid at room temperature, when liquid it is considered to be an oil. In an embodiment, extracted lipid of the invention has not been blended with another lipid such as DHA not produced by another source (for example, DHA from fish oil). In an embodiment, following extraction the ratio of one or more or all of, oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, and total ω6 fatty acids: total ω3 fatty acids, has not been significantly altered (for example, no greater than a 10% or 5% alteration) when compared to the ratio in the intact seed or cell. In an another embodiment, the extracted plant lipid has not been exposed to a procedure, such as hydrogenation or fractionation, which may alter the ratio of one or more or all of, oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, and total ω6 fatty acids:total ω3 fatty acids, when compared to the ratio in the intact seed or cell. When the extracted plant lipid of the invention is comprised in an oil, the oil may further comprise non-fatty acid molecules such as sterols.

[0463] As used herein, the terms "extracted plant oil" and "isolated plant oil" refer to a substance or composition comprising extracted plant lipid or isolated plant lipid and which is a liquid at room temperature. The oil is obtained from a plant or part thereof such as seed. The extracted or isolated oil can be a relatively crude composition obtained by, for example, crushing a plant seed, or a more purified composition where most, if not all, of one or more or each of the water, nucleic acids, proteins and carbohydrates derived from the plant material have been removed. The composition may comprise other components which may be lipid or non-lipid. In an embodiment, the oil composition comprises at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% (w/w) extracted plant lipid. In an embodiment, extracted oil of the invention has not been blended with another oil such as DHA not produced by another source (for example, DHA from fish oil). In an embodiment, following extraction, the ratio of one or more or all of, oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, and total ω6 fatty acids: total ω3 fatty acids, has not been significantly altered (for example, no greater than a 10% or 5% alteration) when compared to the ratio in the intact seed or cell. In an another embodiment, the extracted plant oil has not been exposed to a procedure, such as hydrogenation or fractionation, which may alter the ratio of one or more or all of, oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, and total ω6 fatty acids:total ω3 fatty acids, when compared to the ratio in the intact seed or cell. Extracted plant oil of the invention may comprise non-fatty acid molecules such as sterols.

[0464] As used herein, an "oil" is a composition comprising predominantly lipid and which is a liquid at room temperature. For instance, oil of the invention preferably comprises at least 75%, at least 80%, at least 85% or at least 90% lipid by weight. Typically, a purified oil comprises at least 90% triacylglycerols (TAG) by weight of the lipid in the oil. Minor components of an oil such as diacylglycerols (DAG), free fatty acids (FFA), phospholipid and sterols may be present as described herein.

[0465] As used herein, the term "fatty acid" refers to a carboxylic acid (or organic acid), often with a long aliphatic tail, either saturated or unsaturated. Typically fatty acids have a carbon-carbon bonded chain of at least 8 carbon atoms in length, more preferably at least 12 carbons in length. Most naturally occurring fatty acids have an even number of carbon atoms because their biosynthesis involves acetate which has two carbon atoms. The fatty acids may be in a free state (non-esterified) or in an esterified form such as part of a triglyceride, diacylglyceride, monoacylglyceride, acyl-CoA (thio-ester) bound or other bound form. The fatty acid may be esterified as a phospholipid such as a phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol forms.

[0466] "Saturated fatty acids" do not contain any double bonds or other functional groups along the chain. The term "saturated" refers to hydrogen, in that all carbons (apart from the carboxylic acid [--COOH] group) contain as many hydrogens as possible. In other words, the omega (ω) end contains 3 hydrogens (CH3-) and each carbon within the chain contains 2 hydrogens (--CH2-).

[0467] "Unsaturated fatty acids" are of similar form to saturated fatty acids, except that one or more alkene functional groups exist along the chain, with each alkene substituting a singly-bonded "--CH2-CH2-" part of the chain with a doubly-bonded "--CH═CH--" portion (that is, a carbon double bonded to another carbon). The two next carbon atoms in the chain that are bound to either side of the double bond can occur in a cis or trans configuration.

[0468] As used herein, the term "monounsaturated fatty acid" refers to a fatty acid which comprises at least 12 carbon atoms in its carbon chain and only one alkene group (carbon-carbon double bond) in the chain. As used herein, the terms "polyunsaturated fatty acid" or "PUFA" refer to a fatty acid which comprises at least 12 carbon atoms in its carbon chain and at least two alkene groups (carbon-carbon double bonds).

[0469] As used herein, the terms "long-chain polyunsaturated fatty acid" and "LC-PUFA" refer to a fatty acid which comprises at least 20 carbon atoms in its carbon chain and at least two carbon-carbon double bonds, and hence include VLC-PUFAs. As used herein, the terms "very long-chain polyunsaturated fatty acid" and "VLC-PUFA" refer to a fatty acid which comprises at least 22 carbon atoms in its carbon chain and at least three carbon-carbon double bonds. Ordinarily, the number of carbon atoms in the carbon chain of the fatty acids refers to an unbranched carbon chain. If the carbon chain is branched, the number of carbon atoms excludes those in sidegroups. In one embodiment, the long-chain polyunsaturated fatty acid is an ω3 fatty acid, that is, having a desaturation (carbon-carbon double bond) in the third carbon-carbon bond from the methyl end of the fatty acid. In another embodiment, the long-chain polyunsaturated fatty acid is an ω6 fatty acid, that is, having a desaturation (carbon-carbon double bond) in the sixth carbon-carbon bond from the methyl end of the fatty acid. In a further embodiment, the long-chain polyunsaturated fatty acid is selected from the group consisting of; arachidonic acid (ARA, 20:4Δ5, 8, 11, 14; ω6), eicosatetraenoic acid (ETA, 20:4Δ8, 11, 14, 17, ω3), eicosapentaenoic acid (EPA, 20:5Δ5, 8, 11, 14, 17; ω3), docosapentaenoic acid (DPA, 22:5Δ7, 10, 13, 16, 19, ω3), or docosahexaenoic acid (DHA, 22:6Δ4, 7, 10, 13, 16, 19, ω3). The LC-PUFA may also be dihomo-γ-linoleic acid (DGLA) or eicosatrienoic acid (ETrA, 20:3Δ11, 14, 17, ω3). It would readily be apparent that the LC-PUFA that is produced according to the invention may be a mixture of any or all of the above and may include other LC-PUFA or derivatives of any of these LC-PUFA. In a preferred embodiment, the ω3 fatty acids are at least DHA, preferably, DPA and DHA, or EPA, DPA and DHA.

[0470] Furthermore, as used herein the terms "long-chain polyunsaturated fatty acid" and "very long-chain polyunsaturated fatty acid" refer to the fatty acid being in a free state (non-esterified) or in an esterified form such as part of a triglyceride, diacylglyceride, monoacylglyceride, acyl-CoA bound or other bound form. The fatty acid may be esterified as a phospholipid such as a phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol forms. Thus, the LC-PUFA may be present as a mixture of forms in the lipid of a cell or a purified oil or lipid extracted from cells, tissues or organisms. In preferred embodiments, the invention provides oil comprising at least 75% or at least 85% triacylglycerols, with the remainder present as other forms of lipid such as those mentioned, with at least said triacylglycerols comprising the LC-PUFA. The oil may subsequently be further purified or treated, for example by hydrolysis with a strong base to release the free fatty acids, or by distillation or the like.

[0471] As used herein, "total ω6 fatty acids" or "total ω6 fatty acid content" or the like refers to the sum of all the ω6 fatty acids, esterified and non-esterified, in the extracted lipid, oil, recombinant cell, plant part or seed, as the context determines, expressed as a percentage of the total fatty acid content. These ω6 fatty acids include (if present) LA, GLA, DGLA, ARA, EDA and ω6-DPA, and exclude any ω3 fatty acids and monounsaturated fatty acids.

[0472] As used herein, "new ω6 fatty acids" or "new ω6 fatty acid content" or the like refers to the sum of all the ω6 fatty acids excluding LA, esterified and non-esterified, in the extracted lipid, oil, recombinant cell, plant part or seed, as the context determines, expressed as a percentage of the total fatty acid content. These new ω6 fatty acids are the fatty acids that are produced in the cells, plants, plant parts and seeds of the invention by the expression of the genetic constructs (exogenous polynucleotides) introduced into the cells, and include (if present) GLA, DGLA, ARA, EDA and ω6-DPA, but exclude LA and any ω3 fatty acids and monounsaturated fatty acids. Exemplary total ω6 fatty acid contents and new ω6 fatty acid contents are determined by conversion of fatty acids in a sample to FAME and analysis by GC, as described in Example 1.

[0473] As used herein, "total ω3 fatty acids" or "total ω3 fatty acid content" or the like refers to the sum of all the ω3 fatty acids, esterified and non-esterified, in the extracted lipid, oil, recombinant cell, plant part or seed, as the context determines, expressed as a percentage of the total fatty acid content. These ω3 fatty acids include (if present) ALA, SDA, ETrA, ETA, EPA, DPA and DHA, and exclude any ω6 fatty acids and monounsaturated fatty acids.

[0474] As used herein, "new ω3 fatty acids" or "new ω3 fatty acid content" or the like refers to the sum of all the ω3 fatty acids excluding ALA, esterified and non-esterified, in the extracted lipid, oil, recombinant cell, plant part or seed, as the context determines, expressed as a percentage of the total fatty acid content. These new ω3 fatty acids are the fatty acids that are produced in the cells, plants, plant parts and seeds of the invention by the expression of the genetic constructs (exogenous polynucleotides) introduced into the cells, and include (if present) SDA, ETrA, ETA, EPA, DPA and DHA, but exclude ALA and any ω6 fatty acids and monounsaturated fatty acids. Exemplary total ω3 fatty acid contents and new ω3 fatty acid contents are determined by conversion of fatty acids in a sample to FAME and analysis by GC, as described in Example 1.

[0475] The desaturase, elongase and acyl transferase proteins and genes encoding them that may be used in the invention are any of those known in the art or homologues or derivatives thereof. Examples of such genes and encoded protein sizes are listed in Table 1. The desaturase enzymes that have been shown to participate in LC-PUFA biosynthesis all belong to the group of so-called "front-end" desaturases.

[0476] As used herein, the term "front-end desaturase" refers to a member of a class of enzymes that introduce a double bond between the carboxyl group and a pre-existing unsaturated part of the acyl chain of lipids, which are characterized structurally by the presence of an N-terminal cytochrome b5 domain, along with a typical fatty acid desaturase domain that includes three highly conserved histidine boxes (Napier et al., 1997).

[0477] Activity of any of the elongases or desaturases for use in the invention may be tested by expressing a gene encoding the enzyme in a cell such as, for example, a yeast cell, a plant cell or preferably in somatic embryos or transgenic plants, and determining whether the cell, embryo or plant has an increased capacity to produce LC-PUFA compared to a comparable cell, embryo or plant in which the enzyme is not expressed.

[0478] In one embodiment one or more of the desaturases and/or elongases for use in the invention can purified from a microalga, i.e. is identical in amino acid sequence to a polypeptide which can be purified from a microalga.

[0479] Whilst certain enzymes are specifically described herein as "bifunctional", the absence of such a term does not necessarily imply that a particular enzyme does not possess an activity other than that specifically defined.

Desaturases

[0480] As used herein, the term "desaturase" refers to an enzyme which is capable of introducing a carbon-carbon double bond into the acyl group of a fatty acid substrate which is typically in an esterified form such as, for example, acyl-CoA esters. The acyl group may be esterified to a phospholipid such as phosphatidylcholine (PC), or to acyl carrier protein (ACP), or in a preferred embodiment to CoA. Desaturases generally may be categorized into three groups accordingly. In one embodiment, the desaturase is a front-end desaturase.

[0481] As used herein, a "Δ4-desaturase" refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 4^th carbon-carbon bond from the carboxyl end of a fatty acid substrate. The "Δ4-desaturase" is at least capable of converting DPA to DHA. The desaturation step to produce DHA from DPA is catalysed by a Δ4-desaturase in organisms other than mammals, and a gene encoding this enzyme has been isolated from the freshwater protist species Euglena gracilis and the marine species Thraustochytriumn sp. (Qiu et al., 2001; Meyer et al., 2003). In one embodiment, the Δ4-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:41, or a Thraustochytrium sp. Δ4-desaturase, a biologically active fragment thereof, or an amino acid sequence which is at least 80% identical to SEQ ID NO:41.

TABLE-US-00001 TABLE 1 Cloned genes involved in LC-PUFA biosynthesis Protein size Enzyme Type of organism Species Accession Nos. (aa's) References Δ4- Protist Euglena gracilis AY278558 541 Meyer et al., 2003 desaturase Algae Pavlova lutherii AY332747 445 Tonon et al., 2003 Isochrysis galbana AAV33631 433 Pereira et al., 2004b Pavlova salina AAY15136 447 Zhou et al., 2007 Thraustochytrid Thraustochytrium aureum AAN75707 515 N/A AAN75708 AAN75709 AAN75710 Thraustochytrium sp. AAM09688 519 Qiu et al. 2001 ATCC21685 Δ5- Mammals Homo sapiens AF199596 444 Cho et al., 1999b desaturase Leonard et al., 2000b Nematode Caenorhabditis elegans AF11440, 447 Michaelson et al., 1998b; NM_069350 Watts and Browse, 1999b Fungi Mortierella alpina AF067654 446 Michaelson et al., 1998a; Knutzon et al., 1998 Pythium irregulare AF419297 456 Hong et al., 2002a Dictyostelium discoideum AB022097 467 Saito et al., 2000 Saprolegnia diclina 470 WO02081668 Diatom Phaeodactylum tricornutum AY082392 469 Domergue et al., 2002 Algae Thraustochytrium sp AF489588 439 Qiu et al., 2001 Thraustochytrium aureum 439 WO02081668 Isochrysis galbana 442 WO02081668 Moss Marchantia polymorpha AY583465 484 Kajikawa et al., 2004 Δ6-desaturase Mammals Homo sapiens NM_013402 444 Cho et al., 1999a; Leonard et al., 2000 Mus musculus NM_019699 444 Cho et al., 1999a Nematode Caenorhabditis elegans Z70271 443 Napier et al., 1998 Plants Borago officinales U79010 448 Sayanova et al., 1997 Echium AY055117 Garcia-Maroto et al., 2002 AY055118 Primula vialii AY234127 453 Sayanova et al., 2003 Anemone leveillei AF536525 446 Whitney et al., 2003 Mosses Ceratodon purpureus AJ250735 520 Sperling et al., 2000 Marchantia polymorpha AY583463 481 Kajikawa et al., 2004 Physcomitrella patens CAA11033 525 Girke et al., 1998 Fungi Mortierella alpina AF110510 457 Huang et al., 1999; AB020032 Sakuradani et al., 1999 Pythium irregulare AF419296 459 Hong et al., 2002a Mucor circinelloides AB052086 467 NCBI* Rhizopus sp. AY320288 458 Zhang et al., 2004 Saprolegnia diclina 453 WO02081668 Diatom Phaeodactylum tricornutum AY082393 477 Domergue et al., 2002 Bacteria Synechocystis L11421 359 Reddy et al., 1993 Algae Thraustochytrium aureum 456 WO02081668 Bifunctional Fish Danio rerio AF309556 444 Hastings et al., 2001 Δ5/Δ6- desaturase C20 Δ8- Algae Euglena gracilis AF139720 419 Wallis and Browse, 1999 desaturase Plants Borago officinales AAG43277 446 Sperling et al., 2001 Δ6-elongase Nematode Caenorhabditis elegans NM_069288 288 Beaudoin et al., 2000 Mosses Physcomitrella patens AF428243 290 Zank et al., 2002 Marchantia polymorpha AY583464 290 Kajikawa et al., 2004 Fungi Mortierella alpina AF206662 318 Parker-Barnes et al., 2000 Algae Pavlova lutheri** 501 WO 03078639 Thraustochytrium AX951565 271 WO 03093482 Thraustochytrium sp** AX214454 271 WO 0159128 PUFA- Mammals Homo sapiens AF231981 299 Leonard et al., 2000b; elongase Leonard et al., 2002 Rattus norvegicus AB071985 299 Inagaki et al., 2002 Rattus norvegicus** AB071986 267 Inagaki et al., 2002 Mus musculus AF170907 279 Tvrdik et al., 2000 Mus musculus AF170908 292 Tvrdik et al., 2000 Fish Danio rerio AF532782 291 (282) Agaba et al., 2004 Danio rerio** NM_199532 266 Lo et al., 2003 Worm Caenorhabditis elegans Z68749 309 Abbott et al., 1998 Beaudoin et al., 2000 Algae Thraustochytrium aureum** AX464802 272 WO 0208401-A2 Pavlova lutheri** 320 WO 03078639 Δ9-elongase Algae Isochrysis galbana AF390174 263 Qi et al., 2002 Euglena gracilis 258 WO 08/128241 Δ5-elongase Algae Ostreococcus tauri AAV67798 300 Meyer et al., 2004 Pyramimonas cordata 268 WO 2010/057246 Pavlova sp. CCMP459 AAV33630 277 Pereira et al., 2004b Pavlova salina AAY15135 302 Robert et al., 2009 Diatom Thalassiosira pseudonana AAV67800 358 Meyer et al., 2004 Fish Oncorhynchus mykiss CAM55862 295 WO 06/008099 Moss Marchantia polymorpha BAE71129 348 Kajikawa et al., 2006 *http://www.ncbi.nlm.nih.gov/ **Function not proven/not demonstrated

[0482] As used herein, a "Δ5-desaturase" refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 5^th carbon-carbon bond from the carboxyl end of a fatty acid substrate. Examples of Δ5-desaturases are listed in Ruiz-Lopez et al. (2012) and Petrie et al. (2010a) and in Table 1 herein. In one embodiment, the Δ8-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:30, a biologically active fragment thereof, or an amino acid sequence which is at least 80% identical to SEQ ID NO:30. In another embodiment, the Δ5-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:32, a biologically active fragment thereof, or an amino acid sequence which is at least 53% identical to SEQ ID NO:32. In another embodiment, the Δ5-desaturase is from Thraustochytrium sp or Emiliania huxleyi.

[0483] As used herein, a "Δ6-desaturase" refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 6^th carbon-carbon bond from the carboxyl end of a fatty acid substrate. Examples of Δ6-desaturases are listed in Ruiz-Lopez et al. (2012) and Petrie et al. (2010a) and in Table 1 herein. Preferred Δ6-desaturases are from Micromonas pusilla, Pythium irregulare or Ostreococus taurii.

[0484] In an embodiment, the Δ6-desaturase is further characterised by having at least two, preferably all three and preferably in a plant cell, of the following: i) greater Δ6-desaturase activity on α-linolenic acid (ALA, 18:3Δ9, 12, 15, ω3) than linoleic acid (LA, 18:2Δ9, 12, ω6) as fatty acid substrate; ii) greater Δ6-desaturase activity on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate; and iii) Δ8-desaturase activity on ETrA. Examples of such Δ6-desaturases are provided in Table 2.

[0485] In an embodiment the Δ6-desaturase has greater activity on an ω3 substrate than the corresponding ω6 substrate and has activity on ALA to produce octadecatetraenoic acid (stearidonic acid, SDA, 18:4Δ6, 9, 12, 15, ω3) with an efficiency of at least 30%, more preferably at least 40%, or most preferably at least 50% when expressed from an exogenous polynucleotide in a recombinant cell such as a plant cell, or at least 35% when expressed in a yeast cell. In one embodiment, the Δ6-desaturase has greater activity, for example, at least about a 2-fold greater Δ6-desaturase activity, on ALA than LA as fatty acid substrate. In another embodiment, the Δ6-desaturase has greater activity, for example, at least about 5 fold greater Δ6-desaturase activity or at least 10-fold greater activity, on ALA-CoA as fatty acid substrate than on ALA joined to the sn-2 position of PC as fatty acid substrate. In a further embodiment, the Δ6-desaturase has activity on both fatty acid substrates ALA-CoA and on ALA joined to the sn-2 position of PC.

TABLE-US-00002 TABLE 2 Desaturases demonstrated to have activity on an acyl-CoA substrate Protein Type of Accession size Enzyme organism Species Nos. (aa's) References Δ6-desaturase Algae Mantoniella CAQ30479 449 Hoffmann et al., 2008 squamata Ostreococcus AAW70159 456 Domergue et al., 2005 tauri Micromonas EEH58637 Petrie et al., 2010a pusilla (SEQ ID NO: 13) Δ5-desaturase Algae Mantoniella CAQ30478 482 Hoffmann et al., 2008 squamata Plant Anemone N/A Sayanova et al., 2007 leveillei ω3-desaturase Fungi Pythium FW362186.1 359 Xue et al., 2012; aphanidermatum WO2008/054565 Fungi Phytophthora FW362214.1 363 Xue et al., 2012; (oomycete) sojae WO2008/054565 Fungi Phytophthora FW362213.1 361 Xue et al., 2012; (oomycete) ramorum WO2008/054565

[0486] In one embodiment, the Δ6-desaturase has no detectable Δ5-desaturase activity on ETA. In another embodiment, the Δ6-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:16, SEQ ID NO:19 or SEQ ID NO:20, a biologically active fragment thereof, or an amino acid sequence which is at least 77% identical to SEQ ID NO:16, SEQ ID NO:19 or SEQ ID NO:20. In another embodiment, the Δ6-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:19 or SEQ ID NO:20, a biologically active fragment thereof, or an amino acid sequence which is at least 67% identical to one or both of SEQ ID NO: 19 or SEQ ID NO:20. The Δ6-desaturase may also have Δ8-desaturase activity.

[0487] As used herein, a "Δ8-desaturase" refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 8^th carbon-carbon bond from the carboxyl end of a fatty acid substrate. The Δ8-desaturase is at least capable of converting ETrA to ETA. Examples of Δ8-desaturases are listed in Table 1. In one embodiment, the Δ8-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:52, a biologically active fragment thereof, or an amino acid sequence which is at least 80% identical to SEQ ID NO:52.

[0488] As used herein, an "ω3-desaturase" refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 3rd carbon-carbon bond from the methyl end of a fatty acid substrate. A ω3-desaturase therefore may convert LA to ALA and GLA to SDA (all C18 fatty acids), or DGLA to ETA and/or ARA to EPA (C20 fatty acids). Some ω3-desaturases (group 1) have activity only on C18 substrates, such as plant and cyanobacterial ω3-desaturases. Such ω3-desaturases are also Δ15-desaturases. Other ω3-desaturases have activity on C20 substrates with no activity (group II) or some activity (group III) on C18 substrates. Such ω3-desaturases are also Δ17-desaturases. Preferred o 3-desaturases are group III type which convert LA to ALA, GLA to SDA, DGLA to ETA and ARA to EPA, such as the Pichia pastoris ω3-desaturase (SEQ ID NO: 12). Examples of n3-desaturases include those described by Pereira et al. (2004a) (Saprolegnia diclina ω3-desaturase, group II), Horiguchi et alt (1998), Berberich et al. (1998) and Spychalla et al. (1997) (C. elegans ω3-desaturase, group III). In a preferred embodiment, the ω3-desaturase is a fungal ω3-desaturase. As used herein, a "fungal ω3-desaturase" refers to an ω3-desaturase which is from a fungal source, including an oomycete source, or a variant thereof whose amino acid sequence is at least 95% identical thereto. Genes encoding numerous ω3-desaturases have been isolated from fungal sources such as, for example, from Phytophthora infestans (Accession No. CAJ30870, WO2005083053; SEQ ID NO: 70), Saprolegnia diclina (Accession No. AAR20444, Pereira et al., 2004a & U.S. Pat. No. 7,211,656), Pythium irregulare (WO2008022963, Group II; SEQ ID NO: 72), Mortierella alpina (Sakuradani et al., 2005; Accession No. BAD91495; WO2006019192), Thalassiosira pseudonana (Armbrust et al., 2004; Accession No. XP_--002291057; WO2005012316, SEQ ID NO: 71), Lachancea kluyveri (also known as Saccharomyces kluyveri; Oura et al., 2004; Accession No. AB118663). Xue et al. (2012) describes ω3-desaturases from the oomycetes Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum which were able to efficiently convert ω6 fatty acid substrates to the corresponding ω3 fatty acids, with a preference for C20 substrates, i.e. they had stronger Δ17-desaturase activity than Δ15-desaturase activity. These enzymes lacked Δ12-desaturase activity, but could use fatty acids in both acyl-CoA and phospholipid fraction as substrates.

[0489] In a more preferred embodiment, the fungal ω3-desaturase is the Pichia pastoris (also known as Komagataella pastoris) ω3-desaturase/Δ15-desaturase (Zhang et al., 2008; Accession No. EF116884; SEQ ID NO: 12), or a polypeptide which is at least 95% identical thereto.

[0490] In an embodiment, the ω3-desaturase is at least capable of converting one of ARA to EPA, DGLA to ETA, GLA to SDA, both ARA to EPA and DGLA to ETA, both ARA to EPA and GLA to SDA, or all three of these.

[0491] In one embodiment, the ω3-desaturase has Δ17-desaturase activity on a C20 fatty acid which has at least three carbon-carbon double bonds, preferably ARA. In another embodiment, the ω3-desaturase has Δ15-desaturase activity on a C18 fatty acid which has three carbon-carbon double bonds, preferably GLA. Preferably, both activities are present.

[0492] As used herein, a "Δ12-desaturase" refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 12^th carbon-carbon bond from the carboxyl end of a fatty acid substrate. Δ12-desaturases typically convert either oleoyl-phosphatidylcholine or oleoyl-CoA to linoleoyl-phosphatidylcholine (18:1-PC) or linoleoyl-CoA (18:1-CoA), respectively. The subclass using the PC linked substrate are referred to as phospholipid-dependent Δ12-desaturases, the latter sublclass as acyl-CoA dependent Δ12-desaturases. Plant and fungal Δ12-desaturases are generally of the former sub-class, whereas animal Δ12-desaturases are of the latter subclass, for example the Δ12-desaturases encoded by genes cloned from insects by Zhou et al. (2008). Many other Δ12-desaturase sequences can be easily identified by searching sequence databases.

[0493] As used herein, a "Δ15-desaturase" refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 15^th carbon-carbon bond from the carboxyl end of a fatty acid substrate. Numerous genes encoding Δ15-desaturases have been cloned from plant and fungal species. For example, U.S. Pat. No. 5,952,544 describes nucleic acids encoding plant Δ15-desaturases (FAD3). These enzymes comprise amino acid motifs that were characteristic of plant Δ15-desaturases. WO200114538 describes a gene encoding soybean FAD3. Many other Δ15-desaturase sequences can be easily identified by searching sequence databases.

[0494] As used herein, a "Δ17-desaturase" refers to a protein which performs a desaturase reaction that introduces a carbon-carbon double bond at the 17^th carbon-carbon bond from the carboxyl end of a fatty acid substrate. A Δ17-desaturase is also regarded as an ω3-desaturase if it acts on a C20 substrate to introduce a desaturation at the ω3 bond.

[0495] In a preferred embodiment, the Δ12-desaturase and/or Δ15-desaturase is a fungal Δ12-desaturase or fungal Δ15-desaturase. As used herein, a "fungal Δ12-desaturase" or "a fungal Δ15-desaturase" refers to a Δ12-desaturase or Δ15-desaturase which is from a fungal source, including an oomycete source, or a variant thereof whose amino acid sequence is at least 95% identical thereto. Genes encoding numerous desaturases have been isolated from fungal sources. U.S. Pat. No. 7,211,656 describes a Δ12 desaturase from Saprolegnia diclina. WO2009016202 describes fungal desaturases from Helobdella robusta, Laccaria bicolor, Lottia gigantea, Microcoleus chthonoplastes, Monosiga brevicollis, Mycosphaerella fijiensis, Mycospaerella graminicola, Naegleria gruben, Nectria haematococca, Nematostella vectensis, Phycomyces blakesleeanus, Trichoderma resii, Physcomitrella patens, Postia placenta, Selaginella moellendorffii and Microdochium nivale. WO2005/012316 describes a Δ12-desaturase from Thalassiosira pseudonana and other fungi. WO2003/099216 describes genes encoding fungal Δ12-desaturases and Δ15-desaturases isolated from Neurospora crassa, Aspergillus nidulans, Botrytis cinerea and Mortierella alpina. WO2007133425 describes fungal Δ15 desaturases isolated from: Saccharomyces kluyveri, Mortierella alpina, Aspergillus nidulans, Neurospora crassa, Fusarium graminearum, Fusarium monilifbrme and Magnaporthe grisea. A preferred Δ12 desaturase is from Phytophthora sojae (Ruiz-Lopez et al., 2012).

[0496] A distinct subclass of fungal Δ12-desaturases, and of fungal Δ15-desaturases, are the bifunctional fungal Δ12/Δ15-desaturases. Genes encoding these have been cloned from Fusarium monoliforme (Accession No. DQ272516, Damude et al., 2006), Acanthamoeba castellanii (Accession No. EF017656, Sayanova et al., 2006), Perkinsus marinus (WO2007042510), Claviceps purpurea (Accession No. EF536898, Meesapyodsuk et al., 2007) and Coprinus cinereus (Accession No. AF269266, Zhang et al., 2007).

[0497] In another embodiment, the ω3-desaturase has at least some activity on, preferably greater activity on, an acyl-CoA substrate than a corresponding acyl-PC substrate. As used herein, a "corresponding acyl-PC substrate" refers to the fatty acid esterified at the sn-2 position of phosphatidylcholine (PC) where the fatty acid is the same fatty acid as in the acyl-CoA substrate. For example, the acyl-CoA substrate may be ARA-CoA and the corresponding acyl-PC substrate is sn-2 ARA-PC. In an embodiment, the activity is at least two-fold greater. Preferably, the ω3-desaturase has at least some activity on both an acyl-CoA substrate and its corresponding acyl-PC substrate and has activity on both C18 and C20 substrates. Examples of such ω3-desaturases are known amongst the cloned fungal desaturases listed above.

[0498] In a further embodiment, the ω3-desaturase comprises amino acids having a sequence as provided in SEQ ID NO:12, a biologically active fragment thereof, or an amino acid sequence which is at least 60% identical to SEQ ID NO: 12, preferably at least 90% or at least 95% identical to SEQ ID NO:12.

[0499] In yet a further embodiment, a desaturase for use in the present invention has greater activity on an acyl-CoA substrate than a corresponding acyl-PC substrate. In another embodiment, a desaturase for use in the present invention has greater activity on an acyl-PC substrate than a corresponding acyl-CoA substrate, but has some activity on both substrates. As outlined above, a "corresponding acyl-PC substrate" refers to the fatty acid esterified at the sn-2 position of phosphatidylcholine (PC) where the fatty acid is the same fatty acid as in the acyl-CoA substrate. In an embodiment, the greater activity is at least two-fold greater. In an embodiment, the desaturase is a Δ5 or Δ6-desaturase, or an ω3-desaturase, examples of which are provided, but not limited to, those listed in Table 2. To test which substrate a desaturase acts on, namely an acyl-CoA or an acyl-PC substrate, assays can be carried out in yeast cells as described in Domergue et al. (2003) and (2005). Acyl-CoA substrate capability for a desaturase can also be inferred when an elongase, when expressed together with the desturase, has an enzymatic conversion efficiency in plant cells of at least about 90% where the elongase catalyses the elongation of the product of the desaturase. On this basis, the Δ5-desaturase and Δ4-desaturases expressed from the GA7 construct (Examples 2 and 3) and variants therefof (Example 5) are capable of desaturating their respective acyl-CoA substrates, ETA-CoA and DPA-CoA.

Elongases

[0500] Biochemical evidence suggests that the fatty acid elongation consists of 4 steps: condensation, reduction, dehydration and a second reduction. In the context of this invention, an "elongase" refers to the polypeptide that catalyses the condensing step in the presence of the other members of the elongation complex, under suitable physiological conditions. It has been shown that heterologous or homologous expression in a cell of only the condensing component ("elongase") of the elongation protein complex is required for the elongation of the respective acyl chain. Thus, the introduced elongase is able to successfully recruit the reduction and dehydration activities from the transgenic host to carry out successful acyl elongations. The specificity of the elongation reaction with respect to chain length and the degree of desaturation of fatty acid substrates is thought to reside in the condensing component. This component is also thought to be rate limiting in the elongation reaction.

[0501] As used herein, a "Δ5-elongase" is at least capable of converting EPA to DPA. Examples of Δ5-elongases include those disclosed in WO2005/103253. In one embodiment, the Δ5-elongase has activity on EPA to produce DPA with an efficiency of at least 60%, more preferably at least 65%, more preferably at least 70% or most preferably at least 80% or 90%. In a further embodiment, the Δ5-elongase comprises an amino acid sequence as provided in SEQ ID NO:37, a biologically active fragment thereof, or an amino acid sequence which is at least 47% identical to SEQ ID NO:37. In a further embodiment, the Δ6-elongase is from Ostreococcus taurii or Ostreococcus lucimarinus (US2010/088776).

[0502] As used herein, a "Δ6-elongase" is at least capable of converting SDA to ETA. Examples of Δ6-elongases include those listed in Table 1. In one embodiment, the elongase comprises amino acids having a sequence as provided in SEQ ID NO:25, a biologically active fragment thereof (such as the fragment provided as SEQ ID NO:26), or an amino acid sequence which is at least 55% identical to one or both of SEQ ID NO:25 or SEQ ID NO:26. In an embodiment, the Δ6-elongase is from Physcomitrella patens (Zank et al., 2002; Accession No. AF428243) or Thalassiosira pseudonana (Ruiz-Lopez et al., 2012).

[0503] As used herein, a "Δ9-elongase" is at least capable of converting ALA to ETrA. Examples of Δ9-elongases include those listed in Table 1. In one embodiment, the Δ9-elongase comprises amino acids having a sequence as provided in SEQ ID NO:43, a biologically active fragment thereof, or an amino acid sequence which is at least 80% identical to SEQ ID NO:43. In another embodiment, the Δ9-elongase comprises amino acids having a sequence as provided in SEQ ID NO:46, a biologically active fragment thereof, or an amino acid sequence which is at least 81% identical to SEQ ID NO:46. In another embodiment, the Δ9-elongase comprises amino acids having a sequence as provided in SEQ ID NO:48, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:48. In another embodiment, the Δ9-elongase comprises amino acids having a sequence as provided in SEQ ID NO:50, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to SEQ ID NO:50. In a further embodiment, the Δ9-elongase has greater activity on an ω6 substrate than the corresponding ω3 substrate, or the converse.

[0504] As used herein, the term "has greater activity on an ω6 substrate than the corresponding ω3 substrate" refers to the relative activity of the enzyme on substrates that differ by the action of an ω3 desaturase. Preferably, the ω6 substrate is LA and the ω3 substrate is ALA.

[0505] An elongase with Δ6-elongase and Δ9-elongase activity is at least capable of (i) converting SDA to ETA and (ii) converting ALA to ETrA and has greater Δ6-elongase activity than Δ9-elongase activity. In one embodiment, the elongase has an efficiency of conversion on SDA to produce ETA which is at least 50%, more preferably at least 60%, and/or an efficiency of conversion on ALA to produce ETrA which is at least 6% or more preferably at least 9%. In another embodiment, the elongase has at least about 6.5 fold greater Δ6-elongase activity than Δ9-elongase activity. In a further embodiment, the elongase has no detectable Δ5-elongase activity

Other Enzymes

[0506] As used herein, the term "1-acyl-glycerol-3-phosphate acyltransferase" (LPAAT), also termed lysophosphatidic acid-acyltransferase or acylCoA-lysophosphatidate-acyltransferase, refers to a protein which acylates sn-1-acyl-glycerol-3-phosphate (sn-1 G-3-P) at the sn-2 position to form phosphatidic acid (PA). Thus, the term "1-acyl-glycerol-3-phosphate acyltransferase activity" refers to the acylation of (sn-1 G-3-P) at the sn-2 position to produce PA (EC 2.3.1.51). Preferred LPAATs are those that can use a polyunsaturated C22 acyl-CoA as substrate to transfer the polyunsaturated C22 acyl group to the sn-2 position of LPA, forming PA. Such LPAATs are exemplified in Example 13 and can be tested as described therein. In an embodiment, an LPAAT useful for the invention comprises amino acids having a sequence as provided in any one of SEQ ID NOs: 63 to 69, a biologically active fragment thereof, or an amino acid sequence which is at least 40% identical to any one or more of SEQ ID NOs: 63 to 69. In a preferred embodiment, an LPAAT useful for the invention comprises amino acids having a sequence as provided in any one of SEQ ID NOs: 64, 65 and 67, a biologically active fragment thereof, or an amino acid sequence which is at least 40% identical to any one or more of SEQ ID NOs: 64, 65 and 67.

[0507] As used herein, the term "diacylglycerol acyltransferase" (EC 2.3.1.20; DGAT) refers to a protein which transfers a fatty acyl group from acyl-CoA to a diacylglycerol substrate to produce a triacylglycerol. Thus, the term "diacylglycerol acyltransferase activity" refers to the transfer of acyl-CoA to diacylglycerol to produce triacylglycerol. There are three known types of DGAT referred to as DGAT1, DGAT2 and DGAT3 respectively. DGAT1 polypeptides typically have 10 transmembrane domains, DGAT2 typically have 2 transmembrane domains, whilst DGAT3 is typically soluble. Examples of DGAT1 polypeptides include polypeptides encoded by DGAT1 genes from Aspergillus fumigatus (Accession No. XP_--755172), Arabidopsis thaliana (CAB44774), Ricinus communis (AAR11479), Vernicia fordii (ABC94472), Vernonia galamensis (ABV21945, ABV21946), Euonymus alatus (AAV31083), Caenorhabditis elegans (AAF82410), Rattus norvegicus (NP_--445889), Homo sapiens (NP_--036211), as well as variants and/or mutants thereof. Examples of DGAT2 polypeptides include polypeptides encoded by DGAT2 genes from Arabidopsis thaliana (Accession No. NP_--566952), Ricinus communis (AAY16324), Vernicia fordii (ABC94474), Mortierella ramanniana (AAK84179), Homo sapiens (Q96PD7, Q58HT5), Bos taurus (Q70VD8), Mus musculus (AAK84175), Micromonas CCMP1545, as well as variants and/or mutants thereof. Examples of DGAT3 polypeptides include polypeptides encoded by DGAT3 genes from peanut (Arachis hypogaea, Saha, et al., 2006), as well as variants and/or mutants thereof.

Polypeptides/Peptides

[0508] The term "recombinant" in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate, compared to its native state if it is produced naturally. In one embodiment the cell is a cell that does not naturally produce the polypeptide. However, the cell may be a cell which comprises a non-endogenous gene that causes an altered amount of the polypeptide to be produced. A recombinant polypeptide of the invention includes polypeptides in the cell, tissue, organ or organism, or cell-free expression system, in which it is produced i.e. a polypeptide which has not been purified or separated from other components of the transgenic (recombinant) cell in which it was produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.

[0509] The terms "polypeptide" and "protein" are generally used interchangeably.

[0510] A polypeptide or class of polypeptides may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another. The % identity of a polypeptide to a reference amino acid sequence is typically determined by GAP analysis (Needleman and Wunsch, 1970; GCG program) with parameters of a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 15 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns two sequences over their entire length. The polypeptide or class of polypeptides may have the same enzymatic activity as, or a different activity than, or lack the activity of, the reference polypeptide. Preferably, the polypeptide has an enzymatic activity of at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the reference polypeptide.

[0511] As used herein a "biologically active" fragment is a portion of a polypeptide defined herein which maintains a defined activity of a full-length reference polypeptide, for example possessing desaturase and/or elongase activity or other enzyme activity. Biologically active fragments as used herein exclude the full-length polypeptide. Biologically active fragments can be any size portion as long as they maintain the defined activity. Preferably, the biologically active fragment maintains at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the full length protein.

[0512] With regard to a defined polypeptide or enzyme, it will be appreciated that % identity figures higher than those provided herein will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide/enzyme comprises an amino acid sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

[0513] Amino acid sequence variants/mutants of the polypeptides of the defined herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide. Such variants/mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired enzyme activity.

[0514] Mutant (altered) peptides can be prepared using any technique known in the art. For example, a polynucleotide defined herein can be subjected to in vitro mutagenesis or DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they possess, for example, desaturase or elongase activity.

[0515] In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

[0516] Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.

[0517] Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites which are not conserved amongst naturally occurring desaturases or elongases. These sites are preferably substituted in a relatively conservative manner in order to maintain enzyme activity. Such conservative substitutions are shown in Table 3 under the heading of "exemplary substitutions".

[0518] In a preferred embodiment a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in Table 3. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell.

[0519] Polypeptides can be produced in a variety of ways, including production and recovery of natural polypeptides or recombinant polypeptides according to methods known in the art. In one embodiment, a recombinant polypeptide is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, such as a host cell defined herein. A more preferred cell to produce the polypeptide is a cell in a plant, especially in a seed in a plant.

Polynucleotides

[0520] The invention also provides and/or uses polynucleotides which may be, for example, a gene, an isolated polynucleotide, a chimeric genetic construct such as a T-DNA molecule, or a chimeric DNA. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein or other materials to perform a particular activity defined herein. The term "polynucleotide" is used interchangeably herein with the term "nucleic acid molecule". By "isolated polynucleotide" we mean a polynucleotide which, if obtained from a natural source, has been separated from the polynucleotide sequences with which it is associated or linked in its native state, or a non-naturally occurring polynucleotide. Preferably, the isolated polynucleotide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated.

TABLE-US-00003 TABLE 3 Exemplary substitutions. Original Exemplary Residue Substitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; his Asp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, ala His (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; phe Lys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S) thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe, ala

[0521] In an embodiment, a polynucleotide of the invention is non-naturally occurring. Examples of non-naturally occurring polynucleotides include, but are not limited to, those that have been mutated (such as by using methods described herein), and polynucleotides where an open reading frame encoding a protein is operably linked to a promoter to which it is not naturally associated (such as in the constructs described herein).

[0522] As used herein, the term "gene" is to be taken in its broadest context and includes the deoxyribonucleotide sequences comprising the transcribed region and, if translated, the protein coding region, of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of at least about 2 kb on either end and which are involved in expression of the gene. In this regard, the gene includes control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals in which case the gene is referred to as a "chimeric gene". The sequences which are located 5' of the protein coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the protein coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region which may be interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into nuclear RNA (hnRNA). Introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. The term "gene" includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above.

[0523] As used herein, a "chimeric DNA" or "chimeric genetic construct" refers to any DNA molecule that is not a native DNA molecule in its native location, also referred to herein as a "DNA construct". Typically, a chimeric DNA or chimeric gene comprises regulatory and transcribed or protein coding sequences that are not found operably linked together in nature i.e. that are heterologous with respect to each other. Accordingly, a chimeric DNA or chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.

[0524] The term "endogenous" is used herein to refer to a substance that is normally present or produced in, for example, an unmodified plant at the same developmental stage as the plant under investigation. An "endogenous gene" refers to a native gene in its natural location in the genome of an organism. As used herein, "recombinant nucleic acid molecule", "recombinant polynucleotide" or variations thereof refer to a nucleic acid molecule which has been constructed or modified by recombinant DNA technology. The terms "foreign polynucleotide" or "exogenous polynucleotide" or "heterologous polynucleotide" and the like refer to any nucleic acid which is introduced into the genome of a cell by experimental manipulations. Foreign or exogenous genes may be genes that are inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The terms "genetically modified", "transgenic" and variations thereof include introducing genes into cells by transformation or transduction, mutating genes in cells and altering or modulating the regulation of a gene in a cell or organisms to which these acts have been done or their progeny. A "genomic region" as used herein refers to a position within the genome where a transgene, or group of transgenes (also referred to herein as a cluster), have been inserted into a cell, or an ancestor thereof. Such regions only comprise nucleotides that have been incorporated by the intervention of man such as by methods described herein.

[0525] The term "exogenous" in the context of a polynucleotide refers to the polynucleotide when present in a cell in an altered amount compared to its native state. In one embodiment, the cell is a cell that does not naturally comprise the polynucleotide. However, the cell may be a cell which comprises a non-endogenous polynucleotide resulting in an altered amount of production of the encoded polypeptide. An exogenous polynucleotide of the invention includes polynucleotides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is present, and polynucleotides produced in such cells or cell-free systems which are subsequently purified away from at least some other components. The exogenous polynucleotide (nucleic acid) can be a contiguous stretch of nucleotides existing in nature, or comprise two or more contiguous stretches of nucleotides from different sources (naturally occurring and/or synthetic) joined to form a single polynucleotide. Typically such chimeric polynucleotides comprise at least an open reading frame encoding a polypeptide of the invention operably linked to a promoter suitable of driving transcription of the open reading frame in a cell of interest.

[0526] As used herein, the term "different exogenous polynucleotides" or variations thereof means that the nucleotide sequence of each polynucleotide are different by at least one, preferably more, nucleotides. The polynucleotides encode RNAs which may or may not be translated to a protein within the cell. In an example, it is preferred that each polynucleotide encodes a protein with a different activity. In another example, each exogenous polynucleotide is less than 95%, less than 90%, or less than 80% identical to the other exogenous polynucleotides. Preferably, the exogenous polynucleotides encode functional proteins/enzymes. Furthermore, it is preferred that the different exogenous polynucleotides are non-overlapping in that each polynucleotide is a distinct region of the, for example, extrachromosomal transfer nucleic acid which does not overlap with another exogenous polynucleotide. At a minimum, each exogenous polnucleotide has a transcription start and stop site, as well as the designated promoter. An individual exogenous polynucloeotide may or may not comprise introns.

[0527] With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polynucleotide comprises a polynucleotide sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

[0528] A polynucleotide of the present invention may selectively hybridise, under stringent conditions, to a polynucleotide that encodes a polypeptide of the present invention. As used herein, stringent conditions are those that (1) employ during hybridisation a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1% (w/v) bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (2) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42° C. in 0.2×SSC and 0.1% SDS and/or (3) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.

[0529] Polynucleotides of the invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Polynucleotides which have mutations relative to a reference sequence can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis or DNA shuffling on the nucleic acid as described above). It is thus apparent that polynucleotides of the invention can be either from a naturally occurring source or recombinant. Preferred polynucleotides are those which have coding regions that are codon-optimised for translation in plant cells, as is known in the art.

Recombinant Vectors

[0530] One embodiment of the present invention includes a recombinant vector, which comprises at least one polynucleotide molecule defined herein, inserted into any vector capable of delivering the polynucleotide molecule into a host cell. Recombinant vectors include expression vectors. Recombinant vectors contain heterologous polynucleotide sequences, that is, polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules defined herein that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived. The vector can be either RNA or DNA and typically is a plasmid. Plasmid vectors typically include additional nucleic acid sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, or preferably binary vectors containing one or more T-DNA regions. Additional nucleic acid sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes encoded in the nucleic acid construct, and sequences that enhance transformation of prokaryotic and eukaryotic (especially plant) cells. The recombinant vector may comprise more than one polynucleotide defined herein, for example three, four, five or six polynucleotides defined herein in combination, preferably a chimeric genetic construct of the invention, each polynucleotide being operably linked to expression control sequences that are operable in the cell of interest. More than one polynucleotide defined herein, for example 3, 4, 5 or 6 polynucleotides, are preferably covalently joined together in a single recombinant vector, preferably within a single T-DNA molecule, which may then be introduced as a single molecule into a cell to form a recombinant cell according to the invention, and preferably integrated into the genome of the recombinant cell, for example in a transgenic plant. Thereby, the polynucleotides which are so joined will be inherited together as a single genetic locus in progeny of the recombinant cell or plant. The recombinant vector or plant may comprise two or more such recombinant vectors, each containing multiple polynucleotides, for example wherein each recombinant vector comprises 3, 4, 5 or 6 polynucleotides.

[0531] "Operably linked" as used herein refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element (promoter) to a transcribed sequence. For example, a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0532] When there are multiple promoters present, each promoter may independently be the same or different.

[0533] Recombinant molecules such as the chimeric DNAs or genetic constructs may also contain (a) one or more secretory signals which encode signal peptide sequences, to enable an expressed polypeptide defined herein to be secreted from the cell that produces the polypeptide or which provide for localisation of the expressed polypeptide, for example for retention of the polypeptide in the endoplasmic reticulum (ER) in the cell or transfer into a plastid, and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion or localisation of a polypeptide defined herein. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules defined herein.

[0534] To facilitate identification of transformants, the nucleic acid construct desirably comprises a selectable or screenable marker gene as, or in addition to, the foreign or exogenous polynucleotide. By "marker gene" is meant a gene that imparts a distinct phenotype to cells expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not have the marker. A selectable marker gene confers a trait for which one can "select" based on resistance to a selective agent (e.g., a herbicide, antibiotic, radiation, heat, or other treatment damaging to untransformed cells). A screenable marker gene (or reporter gene) confers a trait that one can identify through observation or testing, i.e., by "screening" (e.g., β-glucuronidase, luciferase, GFP or other enzyme activity not present in untransformed cells). The marker gene and the nucleotide sequence of interest do not have to be linked. The actual choice of a marker is not crucial as long as it is functional (i.e., selective) in combination with the cells of choice such as a plant cell.

[0535] Examples of bacterial selectable markers are markers that confer antibiotic resistance such as ampicillin, erythromycin, chloramphenicol or tetracycline resistance, preferably kanamycin resistance. Exemplary selectable markers for selection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin phosphotransferase (nptII) gene conferring resistance to kanamycin, paromomycin, G418; a glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides as, for example, described in EP 256223; a glutamine synthetase gene conferring, upon overexpression, resistance to glutamine synthetase inhibitors such as phosphinothricin as, for example, described in WO 87/05327, an acetyltransferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin as, for example, described in EP 275957, a gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine as, for example, described by Hinchee et al. (1988), a bar gene conferring resistance against bialaphos as, for example, described in WO91/02071; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988); a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate (Thillet et al., 1988); a mutant acetolactate synthase gene (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP 154,204); a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan; or a dalapon dehalogenase gene that confers resistance to the herbicide.

[0536] Preferred screenable markers include, but are not limited to, a uidA gene encoding a β-glucuronidase (GUS) enzyme for which various chromogenic substrates are known, a green fluorescent protein gene (Niedz et al., 1995) or derivatives thereof; a luciferase (luc) gene (Ow et al., 1986), which allows for bioluminescence detection, and others known in the art. By "reporter molecule" as used in the present specification is meant a molecule that, by its chemical nature, provides an analytically identifiable signal that facilitates determination of promoter activity by reference to protein product.

[0537] Preferably, the nucleic acid construct is stably incorporated into the genome of the cell, such as the plant cell. Accordingly, the nucleic acid may comprise appropriate elements which allow the molecule to be incorporated into the genome, preferably the right and left border sequences of a T-DNA molecule, or the construct is placed in an appropriate vector which can be incorporated into a chromosome of the cell.

Expression

[0538] As used herein, an expression vector is a DNA vector that is capable of transforming a host cell and of effecting expression of one or more specified polynucleotide molecule(s). Preferred expression vectors of the present invention can direct gene expression in yeast and/or plant cells. Expression vectors useful for the invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention. In particular, polynucleotides or vectors useful for the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter and enhancer sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. The choice of the regulatory sequences used depends on the target organism such as a plant and/or target organ or tissue of interest. Such regulatory sequences may be obtained from any eukaryotic organism such as plants or plant viruses, or may be chemically synthesized. A variety of such transcription control sequences are known to those skilled in the art. Particularly preferred transcription control sequences are promoters active in directing transcription in plants, either constitutively or stage and/or tissue specific, depending on the use of the plant or parts thereof.

[0539] A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

[0540] A number of constitutive promoters that are active in plant cells have been described. Suitable promoters for constitutive expression in plants include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliform virus promoter, the commelina yellow mottle virus promoter, the light-inducible promoter from the small subunit of the ribulose-1,5-bis-phosphate carboxylase, the rice cytosolic triosephosphate isomerase promoter, the adenine phosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 gene promoter, the mannopine synthase and octopine synthase promoters, the Adh promoter, the sucrose synthase promoter, the R gene complex promoter, and the chlorophyll α/β binding protein gene promoter

[0541] For the purpose of expression in source tissues of the plant, such as the leaf, seed, root or stem, it is preferred that the promoters utilized in the present invention have relatively high expression in these specific tissues. For this purpose, one may choose from a number of promoters for genes with tissue- or cell-specific or -enhanced expression. Examples of such promoters reported in the literature include the chloroplast glutamine synthetase GS2 promoter from pea, the chloroplast fructose-1,6-biphosphatase promoter from wheat, the nuclear photosynthetic ST-LS1 promoter from potato, the serine/threonine kinase promoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana. Also reported to be active in photosynthetically active tissues are ribulose-1,5-bisphosphate carboxylase promoters, and Cab promoters.

[0542] A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals, also can be used for expression of genes in plant cells, including promoters regulated by (1) heat, (2) light (e.g., pea RbcS-3A promoter, maize RbcS promoter); (3) hormones, such as abscisic acid, (4) wounding (e.g., WunI); or (5) chemicals, such as methyl jasmonate, salicylic acid, steroid hormones, alcohol, Safeners (WO97/06269), or it may also be advantageous to employ (6) organ-specific promoters.

[0543] As used herein, the term "plant seed specific promoter" or variations thereof refer to a promoter that preferentially, when compared to other plant tissues, directs gene transcription in a developing seed of a plant. In an embodiment, the seed specific promoter is expressed at least 5-fold more strongly in the developing seed of the plant relative to the leaves and/or stems of the plant, and is preferably expressed more strongly in the embryo of the developing seed compared to other plant tissues. Preferably, the promoter only directs expression of a gene of interest in the developing seed, and/or expression of the gene of interest in other parts of the plant such as leaves is not detectable by Northern blot analysis and/or RT-PCR. Typically, the promoter drives expression of genes during growth and development of the seed, in particular during the phase of synthesis and accumulation of storage compounds in the seed. Such promoters may drive gene expression in the entire plant storage organ or only part thereof such as the seedcoat, or cotyledon(s), preferably in the embryos, in seeds of dicotyledonous plants or the endosperm or aleurone layer of a seeds of monocotyledonous plants.

[0544] Preferred promoters for seed-specific expression include i) promoters from genes encoding enzymes involved in fatty acid biosynthesis and accumulation in seeds, such as desaturases and elongases, ii) promoters from genes encoding seed storage proteins, and iii) promoters from genes encoding enzymes involved in carbohydrate biosynthesis and accumulation in seeds. Seed specific promoters which are suitable are the oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USP promoter (Baumlein et al., 1991), the Arabidopsis oleosin promoter (WO98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter (WO91/13980) or the legumin LeB4 promoter from Vicia faba (Baumlein et al., 1992), and promoters which lead to the seed-specific expression in monocots such as maize, barley, wheat, rye, rice and the like. Notable promoters which are suitable are the barley lpt2 or lpt1 gene promoter (WO95/15389 and WO95/23230) or the promoters described in WO99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene). Other promoters include those described by Broun et al. (1998), Potenza et al. (2004), US20070192902 and US20030159173. In an embodiment, the seed specific promoter is preferentially expressed in defined parts of the seed such as the embryo, cotyledon(s) or the endosperm. Examples of such specific promoters include, but are not limited to, the FP1 promoter (Ellerstrom et al., 1996), the pea legumin promoter (Perrin et al., 2000), the bean phytohemagglutnin promoter (Perrin et al, 2000), the conlinin 1 and conlinin 2 promoters for the genes encoding the flax 2S storage proteins (Cheng et al., 2010), the promoter of the FAE1 gene from Arabidopsis thaliana, the BnGLP promoter of the globulin-like protein gene of Brassica napus, the LPXR promoter of the peroxiredoxin gene from Linum usitatissimurnm.

[0545] The 5' non-translated leader sequence can be derived from the promoter selected to express the heterologous gene sequence of the polynucleotide of the present invention, or preferably is heterologous with respect to the coding region of the enzyme to be produced, and can be specifically modified if desired so as to increase translation of mRNA. For a review of optimizing expression of transgenes, see Koziel et al. (1996). The 5' non-translated regions can also be obtained from plant viral RNAs (Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus, Alfalfa mosaic virus, among others) from suitable eukaryotic genes, plant genes (wheat and maize chlorophyll a/b binding protein gene leader), or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. The leader sequence could also be derived from an unrelated promoter or coding sequence. Leader sequences useful in context of the present invention comprise the maize Hsp70 leader (U.S. Pat. No. 5,362,865 and U.S. Pat. No. 5,859,347), and the TMV omega element.

[0546] The termination of transcription is accomplished by a 3' non-translated DNA sequence operably linked in the chimeric vector to the polynucteotide of interest. The 3' non-translated region of a recombinant DNA molecule contains a polyadenylation signal that functions in plants to cause the addition of adenylate nucleotides to the 3' end of the RNA. The 3' non-translated region can be obtained from various genes that are expressed in plant cells. The nopaline synthase 3' untranslated region, the 3' untranslated region from pea small subunit Rubisco gene, the 3' untranslated region from soybean 7S seed storage protein gene or a flax conlinin gene are commonly used in this capacity. The 3' transcribed, non-translated regions containing the polyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes are also suitable.

[0547] Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules defined herein include, but are not limited to, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of stability sequences to mRNAs, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.

Recombinant Cells

[0548] The invention also provides a recombinant cell, preferably a recombinant plant cell, which is a host cell transformed with one or more recombinant molecules, such as the polynucleotides, chimeric genetic constructs or recombinant vectors defined herein. The recombinant cell may comprise any combination thereof, such as two or three recombinant vectors, or a recombinant vector and one or more additional polynucleotides or chimeric DNAs. Suitable cells of the invention include any cell that can be transformed with a polynucleotide, chimeric DNA or recombinant vector of the invention, such as for example, a molecule encoding a polypeptide or enzyme described herein. The cell is preferably a cell which is thereby capable of being used for producing LC-PUFA. The recombinant cell may be a cell in culture, a cell in vitro, or in an organism such as for example a plant, or in an organ such as for example a seed or a leaf. Preferably, the cell is in a plant or plant part, more preferably in the seed of a plant.

[0549] Host cells into which the polynucleotide(s) are introduced can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule. Such nucleic acid molecules may be related to LC-PUFA synthesis, or unrelated. Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing proteins defined herein, in which case the recombinant cell derived therefrom has an enhanced capability of producing the polypeptides, or can be capable of producing such proteins only after being transformed with at least one polynucleotide of the invention. In an embodiment, a recombinant cell of the invention has a enhanced capacity to synthesize a long chain polyunsaturated fatty acid. As used herein, the term "cell with an enhanced capacity to synthesize a long chain polyunsaturated fatty acid" is a relative term where the recombinant cell of the invention is compared to the host cell lacking the polynucleotide(s) of the invention, with the recombinant cell producing more long chain polyunsaturated fatty acids, or a greater concentration of LC-PUFA such as DHA (relative to other fatty acids), than the native cell. The cell with an enhanced capacity to synthesize another product, such as for example another fatty acid, a lipid, a carbohydrate such as starch, an RNA molecule, a polypeptide, a pharmaceutical or other product has a corresponding meaning.

[0550] Host cells of the present invention can be any cell capable of producing at least one protein described herein, and include bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells. The cells may be prokaryotic or eukaryotic. Preferred host cells are yeast and plant cells. In a preferred embodiment, the plant cell is a seed cell, in particular a cell in a cotyledon or endospermnn of a seed. In one embodiment, the cell is an animal cell or an algal cell. The animal cell may be of any type of animal such as, for example, a non-human animal cell, a non-human vertebrate cell, a non-human mammalian cell, or cells of aquatic animals such as, fish or crustacea, invertebrates, insects, etc. The cells may be of an organism suitable for a fermentation process. As used herein, the term the "fermentation process" refers to any fermentation process or any process comprising a fermentation step. Examples of fermenting microorganisms include fungal organisms, such as yeast. As used herein, "yeast" includes Saccharomyces spp., Saccharomyces cerevisiae, Saccharomyces carlbergensis, Candida spp., Kluveromyces spp., Pichia spp., Hansenula spp., Trichoderma spp., Lipomyces starkey, and Yarrowia lipolytica. Preferred yeast include strains of the Saccharomyces spp., and in particular, Saccharomyces cerevisiae.

Transgenic Plants

[0551] The invention also provides a plant comprising a cell of the invention, such as a transgenic plant comprising one or more polynucleotides of the invention. The term "plant" as used herein as a noun refers to whole plants, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like. The term "plant part" refers to all plant parts that comprise the plant DNA, including vegetative structures such as, for example, leaves or stems, roots, floral organs or structures, pollen, seed, seed parts such as an embryo, endosperm, scutellum or seed coat, plant tissue such as, for example, vascular tissue, cells and progeny of the same, as long as the plant part synthesizes lipid according to the invention.

[0552] A "transgenic plant", "genetically modified plant" or variations thereof refers to a plant that contains a gene construct ("transgene") not found in a wild-type plant of the same species, variety or cultivar. Transgenic plants as defined in the context of the present invention include plants and their progeny which have been genetically modified using recombinant techniques to cause production of the lipid or at least one polypeptide defined herein in the desired plant or plant organ. Transgenic plant cells and transgenic plant parts have corresponding meanings. A "transgene" as referred to herein has the normal meaning in the art of biotechnology and includes a genetic sequence which has been produced or altered by recombinant DNA or RNA technology and which has been introduced into a cell of the invention, preferably a plant cell. The transgene may include genetic sequences derived from a plant cell which may be of the same species, variety or cultivar as the plant cell into which the transgene is introduced or of a different species, variety or cultivar, or from a cell other than a plant cell. Typically, the transgene has been introduced into the cell, such as a plant, by human manipulation such as, for example, by transformation but any method can be used as one of skill in the art recognizes.

[0553] The terms "seed" and "grain" are used interchangeably herein. "Grain" refers to mature grain such as harvested grain or grain which is still on a plant but ready for harvesting, but can also refer to grain after imbibition or germination, according to the context. Mature grain or seed commonly has a moisture content of less than about 18-20%. "Developing seed" as used herein refers to a seed prior to maturity, typically found in the reproductive structures of the plant after fertilisation or anthesis, but can also refer to such seeds prior to maturity which are isolated from a plant.

[0554] As used herein, the term "obtaining a plant part" or "obtaining a seed" refers to any means of obtaining a plant part or seed, respectively, including harvesting of the plant parts or seed from plants in the field or in containment such as a greenhouse or growth chamber, or by purchase or receipt from a supplier of the plant parts or seed. The seed may be suitable for planting i.e. able to germinate and produce progeny plants, or alternatively has been processed in such a way that it is no longer able to germinate, e.g. cracked, polished or milled seed which is useful for food or feed applications, or for extraction of lipid of the invention.

[0555] As used herein, the term "plant storage organ" refers to a part of a plant specialized to storage energy in the form of, for example, proteins, carbohydrates, fatty acids and/or oils. Examples of plant storage organs are seed, fruit, tuberous roots, and tubers. A preferred plant storage organ of the invention is seed.

[0556] As used herein, the term "phenotypically normal" refers to a genetically modified plant or plant organ, particularly a storage organ such as a seed, tuber or fruit of the invention not having a significantly reduced ability to grow and reproduce when compared to an unmodified plant or plant organ. In an embodiment, the genetically modified plant or plant organ which is phenotypically normal comprises an exogenous polynucleotide encoding a silencing suppressor operably linked to a plant storage organ specific promoter and has an ability to grow or reproduce which is essentially the same as an isogenic plant or organ not comprising said polynucleotide. Preferably, the biomass, growth rate, germination rate, storage organ size, seed size and/or the number of viable seeds produced is not less than 90% of that of a plant lacking said exogenous polynucleotide when grown under identical conditions. This term does not encompass features of the plant which may be different to the wild-type plant but which do not effect the usefulness of the plant for commercial purposes such as, for example, a ballerina phenotype of seedling leaves.

[0557] Plants provided by or contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. In preferred embodiments, the plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, or pea), or other legumes. The plants may be grown for production of edible roots, tubers, leaves, stems, flowers or fruit. The plants may be vegetables or ornamental plants. The plants of the invention may be: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), mustard (Brassica juncea), flax (Linum usitatissimum), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolour, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citrus tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, or barley.

[0558] In a preferred embodiment, the plant is an angiosperm.

[0559] In an embodiment, the plant is an oilseed plant, preferably an oilseed crop plant. As used herein, an "oilseed plant" is a plant species used for the commercial production of oils from the seeds of the plant. The oilseed plant may be oil-seed rape (such as canola), maize, sunflower, soybean, sorghum, flax (linseed) or sugar beet. Furthermore, the oilseed plant may be other Brassicas, cotton, peanut, poppy, mustard, castor bean, sesame, safflower, or nut producing plants. The plant may produce high levels of oil in its fruit, such as olive, oil palm or coconut. Horticultural plants to which the present invention may be applied are lettuce, endive, or vegetable brassicas including cabbage, broccoli, or cauliflower. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, tomato, or pepper.

[0560] In a further preferred embodiment, the non-transgenic plant used to produce a transgenic plant of the invention produces oil, especially in the seed, which has i) less than 20%, less than 10% or less than 5% 18:2 fatty acids and/or ii) less than 10% or less than 5% 18:3 fatty acids.

[0561] In a preferred embodiment, the transgenic plant is homozygous for each and every gene that has been introduced (transgene) so that its progeny do not segregate for the desired phenotype. The transgenic plant may also be heterozygous for the introduced transgene(s), preferably uniformly heterozygous for the transgene, such as for example in F1 progeny which have been grown from hybrid seed. Such plants may provide advantages such as hybrid vigour, well known in the art.

[0562] Where relevant, the transgenic plants may also comprise additional transgenes encoding enzymes involved in the production of LC-PUFAs such as, but not limited to, a Δ6-desaturase, a Δ9-elongase, a Δ8-desaturase, a Δ6-elongase, a Δ5-desaturase, an ω3-desaturase, a Δ4-desaturase, a Δ5-elongase, diacylglycerol acyltransferase, LPAAT, a Δ17-desaturase, a Δ15-desaturase and/or a Δ12 desaturase. Examples of such enzymes with one of more of these activities are known in the art and include those described herein. In specific examples, the transgenic plant at least comprises exogenous polynucleotides encoding;

[0563] a) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase and a Δ6-elongase,

[0564] b) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase and a Δ9-elongase,

[0565] c) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase, a Δ6-elongase, and a Δ15-desaturase,

[0566] d) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase, a Δ9-elongase, and a Δ15-desaturase,

[0567] e) a Δ4-desaturase, a Δ5-desaturase, a Δ6-desaturase, a Δ5-elongase, a Δ6-elongase, and a Δ17-desaturase, or

[0568] f) a Δ4-desaturase, a Δ5-desaturase, a Δ8-desaturase, a Δ5-elongase, a Δ9-elongase, and a Δ17-desaturase.

[0569] In an embodiment, the exogenous polynucleotides encode set of polypeptides which are a Pythium irregulare Δ6-desaturase, a Thraustochytrid Δ5-desaturase or an Emiliana huxleyi Δ8-desaturase, a Physcomitrella patens Δ6-elongase, a Thraustochytrid Δ5-elongase or an Ostreocccus taurii Δ5-elongase, a Phytophthora infestans ω3-desaturase or a Pythium irregulare ω3-desaturase, and a Thraustochytrid Δ4-desaturase.

[0570] In an embodiment, plants of the invention are grown in the field, preferably as a population of at least 1,000 or 1,000,000 plants that are essentially the same, or in an area of at least 1 hectare. Planting densities differ according to the plant species, plant variety, climate, soil conditions, fertiliser rates and other factors as known in the art. For example, canola is typically grown at a planting density of 1.2-1.5 million plants per hectare. Plants are harvested as is known in the art, which may comprise swathing, windrowing and/or reaping of plants, followed by threshing and/or winnowing of the plant material to separate the seed from the remainder of the plant parts often in the form of chaff. Alternatively, seed may be harvested from plants in the field in a single process, namely combining.

Transformation of Plants

[0571] Transgenic plants can be produced using techniques known in the art, such as those generally described in A. Slater et al., Plant Biotechnology--The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).

[0572] As used herein, the terms "stably transforming", "stably transformed" and variations thereof refer to the integration of the exogenous nucleic acid molecules into the genome of the cell such that they are transferred to progeny cells during cell division without the need for positively selecting for their presence. Stable transformants, or progeny thereof, can be selected by any means known in the art such as Southern blots on chromosomal DNA or in situ hybridization of genomic DNA.

[0573] Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because DNA can be introduced into cells in whole plant tissues or plant organs or explants in tissue culture, for either transient expression or for stable integration of the DNA in the plant cell genome. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat. No. 5,104,310, U.S. Pat. No. 5,004,863 or U.S. Pat. No. 5,159,135) including floral dipping methods using Agrobacterium or other bacteria that can transfer DNA into plant cells. The region of DNA to be transferred is defined by the border sequences, and the intervening DNA (T-DNA) is usually inserted into the plant genome. Further, the integration of the T-DNA is a relatively precise process resulting in few rearrangements. In those plant varieties where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer. Preferred Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., In: Plant DNA Infectious Agents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203 (1985).

[0574] Acceleration methods that may be used include, for example, microprojectile bombardment and the like. One example of a method for delivering transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method has been reviewed by Yang et al., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994). Non-biological particles (microprojectiles) that may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like. A particular advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly transforming monocots, is that neither the isolation of protoplasts, nor the susceptibility of Agrobacterium infection are required.

[0575] In another alternative embodiment, plastids can be stably transformed. Methods disclosed for plastid transformation in higher plants include particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination (U.S. Pat. No. 5,451,513, U.S. Pat. No. 5,545,818, U.S. Pat. No. 5,877,402, U.S. Pat. No. 5,932,479, and WO99/05265).

[0576] Other methods of cell transformation can also be used and include but are not limited to introduction of DNA into plants by direct DNA transfer into pollen, by direct injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos.

[0577] The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., (1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.

[0578] The development or regeneration of plants containing the foreign, exogenous gene is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired exogenous nucleic acid is cultivated using methods well known to one skilled in the art.

[0579] To confirm the presence of the transgenes in transgenic cells and plants, a polymerase chain reaction (PCR) amplification or Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts, may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.

[0580] A transgenic plant formed using Agrobacterium or other transformation methods typically contains a single genetic locus on one chromosome. Such transgenic plants can be referred to as being hemizygous for the added gene(s). More preferred is a transgenic plant that is homozygous for the added gene(s); i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by self-fertilising a hemizygous transgenic plant, germinating some of the seed produced and analyzing the resulting plants for the gene of interest.

[0581] It is also to be understood that two different transgenic plants that contain two independently segregating exogenous genes or loci can also be crossed (mated) to produce offspring that contain both sets of genes or loci. Selfing of appropriate F1 progeny can produce plants that are homozygous for both exogenous genes or loci. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in Fehr, In: Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Enhancing Exogenous RNA Levels and Stabilized Expression

Silencing Suppressors

[0582] In an embodiment, a cell, plant or plant part of the invention comprises an exogenous polynucleotide encoding a silencing suppressor protein.

[0583] Post-transcriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs for degradation PTGS occurs in plants or fungi stably or transiently transformed with foreign (heterologous) or endogenous DNA and results in the reduced accumulation of RNA molecules with sequence similarity to the introduced nucleic acid.

[0584] It has widely been considered that co-expression of a silencing suppressor with a transgene of interest will increase the levels of RNA present in the cell transcribed from the transgene. Whilst this has proven true for cells in vitro, significant side-effects have been observed in many whole plant co-expression studies. More specifically, as described in Mallory et al. (2002), Chapman et al. (2004), Chen et al. (2004), Dunoyer et al. (2004), Zhang et al. (2006), Lewsey et al. (2007) and Meng et al. (2008) plants expressing silencing suppressors, generally under constitutive promoters, are often phenotypically abnormal to the extent that they are not useful for commercial production.

[0585] Recently, it has been found that RNA molecule levels can be increased, and/or RNA molecule levels stabilized over numerous generations, by limiting the expression of the silencing suppressor to a seed of a plant or part thereof (WO2010/057246). As used herein, a "silencing suppressor protein" or SSP is any polypeptide that can be expressed in a plant cell that enhances the level of expression product from a different transgene in the plant cell, particularly over repeated generations from the initially transformed plant. In an embodiment, the SSP is a viral silencing suppressor or mutant thereof. A large number of viral silencing suppressors are known in the art and include, but are not limited to P19, V2, P38, Pe-Po and RPV-P0. In an embodiment, the viral silencing suppressor comprises amino acids having a sequence as provided in any one of SEQ ID NOs 53 to 57, a biologically active fragment thereof, or an amino acid sequence which is at least 50% identical to any one or more of SEQ ID NOs 53 to 57 and which has activity as a silencing suppressor.

[0586] As used herein, the terms "stabilising expression", "stably expressed", "stabilised expression" and variations thereof refer to level of the RNA molecule being essentially the same or higher in progeny plants over repeated generations, for example at least three, at least five or at least 10 generations, when compared to isogenic plants lacking the exogenous polynucleotide encoding the silencing suppressor. However, this term(s) does not exclude the possibility that over repeated generations there is some loss of levels of the RNA molecule when compared to a previous generation, for example not less than a 10% loss per generation.

[0587] The suppressor can be selected from any source e.g. plant, viral, mammal etc. See WO2010/057246 for a list of viruses from which the suppressor can be obtained and the protein (eg B2, P14 etc) or coding region designation for the suppressor from each particular virus. Multiple copies of a suppressor may be used. Different suppressors may be used together (e.g., in tandem).

RNA Molecules

[0588] Essentially any RNA molecule which is desirable to be expressed in a plant seed can be co-expressed with the silencing suppressor. The encoded polypeptides may be involved in metabolism of oil, starch, carbohydrates, nutrients, etc., or may be responsible for the synthesis of proteins, peptides, fatty acids, lipids, waxes, oils, starches, sugars, carbohydrates, flavors, odors, toxins, carotenoids. hormones, polymers, flavonoids, storage proteins, phenolic acids, alkaloids, lignins, tannins, celluloses, glycoproteins, glycolipids, etc, preferably the biosynthesis or assembly of TAG.

[0589] In a particular example, the plants produced increased levels of enzymes for oil production in plants such as Brassicas, for example canola or sunflower, safflower, flax, cotton, soya bean, Camelina or maize.

Levels of LC-PUFA Produced

[0590] The levels of the LC-PUFA or combination of LC-PUFAs that are produced in the recombinant cell or plant part such as seed are of importance. The levels may be expressed as a composition (in percent) of the total fatty acid that is a particular LC-PUFA or group of related LC-PUFA, for example the ω3 LC-PUFA or the ω6 LC-PUFA, or the VLC-PUFA, or other which may be determined by methods known in the art. The level may also be expressed as a LC-PUFA content, such as for example the percentage of LC-PUFA in the dry weight of material comprising the recombinant cells, for example the percentage of the weight of seed that is LC-PUFA. It will be appreciated that the LC-PUFA that is produced in an oilseed may be considerably higher in terms of LC-PUFA content than in a vegetable or a grain that is not grown for oil production, yet both may have similar LC-PUFA compositions, and both may be used as sources of LC-PUFA for human or animal consumption.

[0591] The levels of LC-PUFA may be determined by any of the methods known in the art. In a preferred method, total lipid is extracted from the cells, tissues or organisms and the fatty acid converted to methyl esters before analysis by gas chromatography (GC). Such techniques are described in Example 1. The peak position in the chromatogram may be used to identify each particular fatty acid, and the area under each peak integrated to determine the amount. As used herein, unless stated to the contrary, the percentage of particular fatty acid in a sample is determined as the area under the peak for that fatty acid as a percentage of the total area for fatty acids in the chromatogram. This corresponds essentially to a weight percentage (w/w). The identity of fatty acids may be confirmed by GC-MS. Total lipid may be separated by techniques known in the art to purify fractions such as the TAG fraction. For example, thin-layer chromatography (TLC) may be performed at an analytical scale to separate TAG from other lipid fractions such as DAG, acyl-CoAs or phospholipid in order to determine the fatty acid composition specifically of TAG.

[0592] In one embodiment, the sum total of ARA, EPA, DPA and DHA in the fatty acids in the extracted lipid is between about 7% and about 25% of the total fatty acids in the cell. In a further embodiment, the total fatty acid in the cell has less than 1% C20:1. In preferred embodiments, the extractable TAG in the cell comprises the fatty acids at the levels referred to herein. Each possible combination of the features defining the lipid as described herein is also encompassed.

[0593] The level of production of LC-PUFA in the recombinant cell, plant or plant part such as seed may also be expressed as a conversion percentage of a specific substrate fatty acid to one or more product fatty acids, which is also referred to herein as a "conversion efficiency" or "enzymatic efficiency". This parameter is based on the fatty acid composition in the lipid extracted from the cell, plant, plant part or seed, i.e., the amount of the LC-PUFA formed (including other LC-PUFA derived therefrom) as a percentage of one or more substrate fatty acids (including all other fatty acids derived therefrom). The general formula for a conversion percentage is: 100× (the sum of percentages of the product LC-PUFA and all products derived therefrom)/(the sum of the percentages of the substrate fatty acid and all products derived therefrom). With regard to DHA, for example, this may be expressed as the ratio of the level of DHA (as a percentage in the total fatty acid content in the lipid) to the level of a substrate fatty acid (e.g. OA, LA, ALA, SDA, ETA or EPA) and all products other than DHA derived from the substrate. The conversion percentage or efficiency of conversion can be expressed for a single enzymatic step in a pathway, or for part or the whole of a pathway.

[0594] Specific conversion efficiencies are calculated herein according to the formulae:

[0595] 1. OA to DHA=100×(% DHA)/(sum % for OA, LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).

[0596] 2. LA to DHA=100×(% DHA)/(sum % for LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).

[0597] 3. ALA to DHA=100×(% DHA)/(sum % for ALA, SDA, ETrA, ETA, EPA, DPA and DHA).

[0598] 4. EPA to DHA=100×(% DHA)/(sum % for EPA, DPA and DHA).

[0599] 5. DPA to DHA (Δ4-desaturase efficiency)=100×(% DHA)/(sum % for DPA and DHA).

[0600] 6. Δ12-desaturase efficiency=100×(sum % for LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA)/(sum % for OA, LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).

[0601] 7. ω3-desaturase efficiency=100×(sum % for ALA, SDA, ETrA, ETA, EPA, DPA and DHA)/(sum % for LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).

[0602] 8. OA to ALA=100×(sum % for ALA, SDA, ETrA, ETA, EPA, DPA and DHA)/(sum % for OA, LA, GLA, DGLA, ARA, EDA, ALA, SDA, ETrA, ETA, EPA, DPA and DHA).

[0603] 9. Δ6-desaturase efficiency (on ω3 substrate ALA)=100×(sum % for SDA, ETA, EPA, DPA and DHA)/(% ALA, SDA, ETrA, ETA, EPA, DPA and DHA).

[0604] 10. Δ6-elongase efficiency (on ω3 substrate SDA)=100×(sum % for ETA, EPA, DPA and DHA)/(sum % for SDA, ETA, EPA, DPA and DHA).

[0605] 11. Δ5-desaturase efficiency (on ω3 substrate ETA)=100×(sum % for EPA, DPA and DHA)/(sum % for ETA, EPA, DPA and DHA).

[0606] 12. Δ5-elongase efficiency (on ω3 substrate EPA)=100×(sum % for DPA and DHA)/(sum % for EPA, DPA and DHA).

[0607] The fatty acid composition of the lipid, preferably seedoil, of the invention, is also characterised by the ratio of ω6 fatty acids:ω3 fatty acids in the total fatty acid content, for either total ω6 fatty acids:total ω3 fatty acids or for new ω6 fatty acids:new ω3 fatty acids. The terms total ω6 fatty acids, total ω3 fatty acids, new ω6 fatty acids and new ω3 fatty acids have the meanings as defined herein. The ratios are calculated from the fatty acid composition in the lipid extracted from the cell, plant, plant part or seed, in the manner as exemplified herein. It is desirable to have a greater level of ω3 than ω6 fatty acids in the lipid, and therefore an ω6:ω3 ratio of less than 1.0 is preferred. A ratio of 0.0 indicates a complete absence of the defined ω6 fatty acids; a ratio of 0.03 was achieved as described in Example 6. Such low ratios can be achieved through the combined use of a Δ6-desaturase which has an ω3 substrate preference together with an ω3-desaturase, particularly a fungal ω3-desaturase such as the Pichia pastoris ω3-desaturase as exemplified herein.

[0608] The yield of LC-PUFA per weight of seed may also be calculated based on the total oil content in the seed and the % DHA in the oil. For example, if the oil content of canola seed is about 40% (w/w) and about 12% of the total fatty acid content of the oil is DHA, the DHA content of the seed is about 4.8% or about 48 mg per gram of seed. As described in Example 2, the DHA content of Arabidopsis seed having about 9% DHA, which has a lower oil content than canola, was about 25 mg/g seed. At a DHA content of about 7%, canola seed or Camelina sativa seed has a DHA content of about 28 mg per grain of seed. The present invention therefore provides Brassica napus, B. juncea and Camelina sativa plants, and seed obtained therefrom, comprising at least about 28 mg DHA per gram seed. The seed has a moisture content as is standard for harvested mature seed after drying down (4-15% moisture). The invention also provides a process for obtaining oil, comprising obtaining the seed and extracting the oil from the seed, and uses of the oil and methods of obtaining the seed comprising harvesting the seeds from the plants according to the invention.

[0609] The amount of DHA produced per hectare can also be calculated if the seed yield per hectare is known or can be estimated. For example, canola in Australia typically yields about 2.5 tonnes seed per hectare, which at 40% oil content yields about 1000 kg of oil. At 12% DHA in the total oil, this provides about 120 kg of DHA per hectare. If the oil content is reduced by 50%, this still provides about 60 kg DHA/ha.

[0610] Evidence to date suggests that some desaturases expressed heterologously in yeast or plants have relatively low activity in combination with some elongases. This may be alleviated by providing a desaturase with the capacity of to use an acyl-CoA form of the fatty acid as a substrate in LC-PUFA synthesis, and this is thought to be advantageous in recombinant cells particularly in plant cells. A particularly advantageous combination for efficient DHA synthesis is a fungal ω3-desaturase, for example such as the Pichia pastoris ω3-desaturase (SEQ ID NO: 12), with a Δ6-desaturase which has a preference for ω3 acyl substrates such as, for example, the Micromonas pusilla Δ6-desaturase (SEQ ID NO: 13), or variants thereof which have at least 95% amino acid sequence identity.

[0611] As used herein, the term "essentially free" means that the composition (for example lipid or oil) comprises little (for example, less than about 0.5%, less than about 0.25%, less than about 0.1%, or less than about 0.01%) or none of the defined component. In an embodiment, "essentially free" means that the component is undetectable using a routine analytical technique, for example a specific fatty acid (such as ω6-docosapentaenoic acid) cannot be detected using gas chromatography as outlined in Example 1.

Production of Oils

[0612] Techniques that are routinely practiced in the art can be used to extract, process, and analyze the oils produced by cells, plants, seeds, etc of the instant invention. Typically, plant seeds are cooked, pressed, and extracted to produce crude oil, which is then degummed, refined, bleached, and deodorized. Generally, techniques for crushing seed are known in the art. For example, oilseeds can be tempered by spraying them with water to raise the moisture content to, e.g., 8.5%, and flaked using a smooth roller with a gap setting of 0.23 to 0.27 mm. Depending on the type of seed, water may not be added prior to crushing. Application of heat deactivates enzymes, facilitates further cell rupturing, coalesces the oil droplets, and agglomerates protein particles, all of which facilitate the extraction process.

[0613] In an embodiment, the majority of the seed oil is released by passage through a screw press. Cakes expelled from the screw press are then solvent extracted, e.g., with hexane, using a heat traced column. Alternatively, crude oil produced by the pressing operation can be passed through a settling tank with a slotted wire drainage top to remove the solids that are expressed with the oil during the pressing operation. The clarified oil can be passed through a plate and frame filter to remove any remaining fine solid particles. If desired, the oil recovered from the extraction process can be combined with the clarified oil to produce a blended crude oil.

[0614] Once the solvent is stripped from the crude oil, the pressed and extracted portions are combined and subjected to normal oil processing procedures. As used herein, the term "purified" when used in connection with lipid or oil of the invention typically means that that the extracted lipid or oil has been subjected to one or more processing steps of increase the purity of the lipid/oil component. For example, a purification step may comprise one or more or all of the group consisting of: degumming, deodorising, decolourising, drying and/or fractionating the extracted oil. However, as used herein, the term "purified" does not include a transesterification process or other process which alters the fatty acid composition of the lipid or oil of the invention so as to increase the DHA content as a percentage of the total fatty acid content. Expressed in other words, the fatty acid composition of the purified lipid or oil is essentially the same as that of the unpurified lipid or oil.

Degumming

[0615] Degumming is an early step in the refining of oils and its primary purpose is the removal of most of the phospholipids from the oil, which may be present as approximately 1-2% of the total extracted lipid. Addition of 2% of water, typically containing phosphoric acid, at 70-80° C. to the crude oil results in the separation of most of the phospholipids accompanied by trace metals and pigments. The insoluble material that is removed is mainly a mixture of phospholipids and triacylglycerols and is also known as lecithin. Degumming can be performed by addition of concentrated phosphoric acid to the crude seedoil to convert non-hydratable phosphatides to a hydratable form, and to chelate minor metals that are present. Gum is separated from the seedoil by centrifugation.

Alkali Refining

[0616] Alkali refining is one of the refining processes for treating crude oil, sometimes also referred to as neutralization. It usually follows degumming and precedes bleaching. Following degumming, the seedoil can treated by the addition of a sufficient amount of an alkali solution to titrate all of the fatty acids and phosphoric acids, and removing the soaps thus formed. Suitable alkaline materials include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate and ammonium hydroxide. This process is typically carried out at room temperature and removes the free fatty acid fraction. Soap is removed by centrifugation or by extraction into a solvent for the soap, and the neutralised oil is washed with water. If required, any excess alkali in the oil may be neutralized with a suitable acid such as hydrochloric acid or sulphuric acid.

Bleaching

[0617] Bleaching is a refining process in which oils are heated at 90-120° C. for 10-30 minutes in the presence of a bleaching earth (0.2-2.0%) and in the absence of oxygen by operating with nitrogen or steam or in a vacuum. This step in oil processing is designed to remove unwanted pigments (carotenoids, chlorophyll, gossypol etc), and the process also removes oxidation products, trace metals, sulphur compounds and traces of soap.

Deodorization

[0618] Deodorization is a treatment of oils and fats at a high temperature (200-260° C.) and low pressure (0.1-1 mm Hg). This is typically achieved by introducing steam into the seedoil at a rate of about 0.1 ml/minute/100 ml of seedoil. After about 30 minutes of sparging, the seedoil is allowed to cool under vacuum. The seedoil is typically transferred to a glass container and flushed with argon before being stored under refrigeration. This treatment improves the colour of the seedoil and removes a majority of the volatile substances or odorous compounds including any remaining free fatty acids, monoacylglycerols and oxidation products.

Winterisation

[0619] Winterization is a process sometimes used in commercial production of oils for the separation of oils and fats into solid (stearin) and liquid (olein) fractions by crystallization at sub-ambient temperatures. It was applied originally to cottonseed oil to produce a solid-free product. It is typically used to decrease the saturated fatty acid content of oils.

Transesterification

[0620] Transesterification is a process that exchanges the fatty acids within and between TAGs or transfers the fatty acids to another alcohol to form an ester, initially by releasing fatty acids from the TAGs either as free fatty acids or as fatty acid esters, usually fatty acid methyl esters or ethyl esters. When combined with a fractionation process, transesterification can be used to modify the fatty acid composition of lipids (Marangoni et al., 1995). Transesterification can use either chemical (e.g. strong acid or base catalysed) or enzymatic means, the latter using lipases which may be position-specific (sn-1/3 or sn-2 specific) for the fatty acid on the TAG, or having a preference for some fatty acids over others (Speranza et al, 2012). The fatty acid fractionation to increase the concentration of LC-PUFA in an oil can be achieved by any of the methods known in the art, such as, for example, freezing crystallization, complex formation using urea, molecular distillation, supercritical fluid extraction and silver ion complexing. Complex formation with urea is a preferred method for its simplicity and efficiency in reducing the level of saturated and monounsaturated fatty acids in the oil (Gamez et al., 2003). Initially, the TAGs of the oil are split into their constituent fatty acids, often in the form of fatty acid esters, by hydrolysis under either acid or base catalysed reaction conditions, whereby one mol of TAG is reacted with at least 3 mol of alcohol (e.g. ethanol for ethyl esters or methanol for methyl esters) with excess alcohol used to enable separation of the formed alkyl esters and the glycerol that is also formed, or by lipases. These free fatty acids or fatty acid esters, which are usually unaltered in fatty acid composition by the treatment, may then be mixed with an ethanolic solution of urea for complex formation. The saturated and monounsaturated fatty acids easily complex with urea and crystallize out on cooling and may subsequently be removed by filtration. The non-urea complexed fraction is thereby enriched with LC-PUFA.

Feedstuffs

[0621] The present invention includes compositions which can be used as feedstuffs. For purposes of the present invention, "feedstuffs" include any food or preparation for human or animal consumption which when taken into the body (a) serve to nourish or build up tissues or supply energy; and/or (b) maintain, restore or support adequate nutritional status or metabolic function. Feedstuffs of the invention include nutritional compositions for babies and/or young children such as, for example, infant formula, and seedmeal of the invention.

[0622] Feedstuffs of the invention comprise, for example, a cell of the invention, a plant of the invention, the plant part of the invention, the seed of the invention, an extract of the invention, the product of the method of the invention, the product of the fermentation process of the invention, or a composition along with a suitable carrier(s). The term "carrier" is used in its broadest sense to encompass any component which may or may not have nutritional value. As the skilled addressee will appreciate, the carrier must be suitable for use (or used in a sufficiently low concentration) in a feedstuff such that it does not have deleterious effect on an organism which consumes the feedstuff.

[0623] The feedstuff of the present invention comprises an oil, fatty acid ester, or fatty acid produced directly or indirectly by use of the methods, cells or plants disclosed herein. The composition may either be in a solid or liquid form. Additionally, the composition may include edible macronutrients, protein, carbohydrate, vitamins, and/or minerals in amounts desired for a particular use. The amounts of these ingredients will vary depending on whether the composition is intended for use with normal individuals or for use with individuals having specialized needs, such as individuals suffering from metabolic disorders and the like.

[0624] Examples of suitable carriers with nutritional value include, but are not limited to, macronutrients such as edible fats, carbohydrates and proteins. Examples of such edible fats include, but are not limited to, coconut oil, borage oil, fungal oil, black current oil, soy oil, and mono- and diglycerides. Examples of such carbohydrates include (but are not limited to): glucose, edible lactose, and hydrolyzed starch. Additionally, examples of proteins which may be utilized in the nutritional composition of the invention include (but are not limited to) soy proteins, electrodialysed whey, electrodialysed skim milk, milk whey, or the hydrolysates of these proteins.

[0625] With respect to vitamins and minerals, the following may be added to the feedstuff compositions of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other such vitamins and minerals may also be added.

[0626] The components utilized in the feedstuff compositions of the present invention can be of semi-purified or purified origin. By semi-purified or purified is meant a material which has been prepared by purification of a natural material or by de novo synthesis.

[0627] A feedstuff composition of the present invention may also be added to food even when supplementation of the diet is not required. For example, the composition may be added to food of any type, including (but not limited to): margarine, modified butter, cheeses, milk, yogurt, chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats, fish and beverages.

[0628] The genus Saccharomyces spp is used in both brewing of beer and wine making and also as an agent in baking, particularly bread. Other yeasts such as oleaginous yeast including, for example, Yarrowia spp, are also useful in LC-PUFA production. Yeasts may be used as an additive in animal feed, such as in aquaculture. It will be apparent that genetically engineered yeast strains can be provided which are adapted to synthesise LC-PUFA as described herein. These yeast strains, or LC-PUFA produced therein, can then be used in food stuffs and in wine and beer making to provide products which have enhanced fatty acid content.

[0629] Additionally, fatty acids produced in accordance with the present invention or host cells transformed to contain and express the subject genes may also be used as animal food supplements to alter an animal's tissue, egg or milk fatty acid composition to one more desirable for human or animal consumption. Examples of such animals include sheep, cattle, horses, poultry such as chickens and the like.

[0630] Furthermore, feedstuffs of the invention can be used in aquaculture to increase the levels of fatty acids in fish or crustaceans such as, for example, prawns for human or animal consumption. Preferred fish are salmon.

[0631] Preferred feedstuffs of the invention are the plants, seed and other plant parts such as leaves and stems which may be used directly as food or feed for humans or other animals. For example, animals may graze directly on such plants grown in the field or be fed more measured amounts in controlled feeding. The invention includes the use of such plants and plant parts as feed for increasing the LC-PUFA levels in humans and other animals.

Compositions

[0632] The present invention also encompasses compositions, particularly pharmaceutical compositions, comprising one or more of the fatty acids and/or resulting oils produced using the methods of the invention.

[0633] A pharmaceutical composition may comprise one or more of the fatty acids and/or oils, in combination with a standard, well-known, non-toxic pharmaceutically-acceptable carrier, adjuvant or vehicle such as phosphate-buffered saline, water, ethanol, polyols, vegetable oils, a wetting agent or an emulsion such as a water/oil emulsion. The composition may be in either a liquid or solid form. For example, the composition may be in the form of a tablet, capsule, ingestible liquid or powder, injectible, or topical ointment or cream. Proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfuming agents.

[0634] Suspensions, in addition to the active compounds, may comprise suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth or mixtures of these substances.

[0635] Solid dosage forms such as tablets and capsules can be prepared using techniques well known in the art. For example, fatty acids produced in accordance with the present invention can be tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch or gelatin, disintegrating agents such as potato starch or alginic acid, and a lubricant such as stearic acid or magnesium stearate. Capsules can be prepared by incorporating these excipients into a gelatin capsule along with antioxidants and the relevant fatty acid(s).

[0636] For intravenous administration, the fatty acids produced in accordance with the present invention or derivatives thereof may be incorporated into commercial formulations.

[0637] A typical dosage of a particular fatty acid is from 0.1 mg to 20 g, taken from one to five times per day (up to 100 g daily) and is preferably in the range of from about 10 mg to about 1, 2, 5, or 10 g daily (taken in one or multiple doses). As known in the art, a minimum of about 300 mg/day of fatty acid, especially LC-PUFA, is desirable. However, it will be appreciated that any amount of fatty acid will be beneficial to the subject.

[0638] Possible routes of administration of the pharmaceutical compositions of the present invention include, for example, enteral (e.g., oral and rectal) and parenteral. For example, a liquid preparation may be administered orally or rectally. Additionally, a homogenous mixture can be completely dispersed in water, admixed under sterile conditions with physiologically acceptable diluents, preservatives, buffers or propellants to form a spray or inhalant.

[0639] The dosage of the composition to be administered to the patient may be determined by one of ordinary skill in the art and depends upon various factors such as weight of the patient, age of the patient, overall health of the patient, past history of the patient, immune status of the patient, etc.

[0640] Additionally, the compositions of the present invention may be utilized for cosmetic purposes. It may be added to pre-existing cosmetic compositions such that a mixture is formed or a fatty acid produced according to the subject invention may be used as the sole "active" ingredient in a cosmetic composition.

EXAMPLES

Example 1

Materials and Methods

Expression of Genes in Plant Cells in a Transient Expression System

[0641] Exogenous genetic constructs were expressed in plant cells in a transient expression system essentially as described by Voinnet et al. (2003) and Wood et al. (2009). Plasmids containing a coding region to be expressed from a strong constitutive promoter such as the CaMV 35S promoter were introduced into Agrobacterium tumefaciens strain AGL1. A chimeric gene 35S:p19 for expression of the p19 viral silencing suppressor was separately introduced into AGL1, as described in WO 2010/057246. The recombinant Agrobacterium cells were grown at 28° C. in LB broth supplemented with 50 mg/L kanamycin and 50 mg/L rifampicin to stationary phase. The bacteria were then pelleted by centrifugation at 5000 g for 15 min at room temperature before being resuspended to OD600=1.0 in an infiltration buffer containing 10 mM MES pH 5.7, 10 mM MgCl₂ and 100 μM acetosyringone. The cells were then incubated at 28° C. with shaking for 3 hours before equal volumes of Agrobacterium cultures containing 35S:p19 and the test chimeric construct(s) of interest were mixed prior to infiltration into leaf tissue. The plants were typically grown for a further five days after infiltration before leaf discs were taken and freeze-dried for GC analysis of the fatty acids.

[0642] Fatty acid methyl esters (FAME) of total leaf lipids in freeze-dried samples were produced by incubating the samples in methanol/HCl/dichloromethane (10/1/1 v/v) solution for 2 hours at 80° C. together with a known amount of hexadecanoic acid as an internal standard. FAMEs were extracted in hexane/DCM, concentrated to a small volume in hexane and injected into a GC. The amount of individual and total fatty acids present in the lipid fractions were quantified on the basis of the known amount of internal standard.

Gas Chromatography (GC) Analysis of Fatty Acids

[0643] FAME were analysed by gas chromatography using an Agilent Technologies 7890A GC (Palo Alto, Calif., USA) equipped with a 30 m SGE-BPX70 column (70% cyanopropyl polysilphenylene-siloxane, 0.25 mm inner diameter, 0.25 mm film thickness), an FID, a split/splitless injector and an Agilent Technologies 7693 Series auto sampler and injector. Helium was used as the carrier gas. Samples were injected in split mode (50:1 ratio) at an oven temperature of 150° C. After injection, the oven temperature was held at 150° C. for 1 min then raised to 210° C. at 3° C. min^-1, again raised to 240° C. at 50° C. mint and finally holding for 1.4 min at 240° C. Peaks were quantified with Agilent Technologies ChemStation software (Rev B.04.03 (16), Palo Alto, Calif., USA) based on the response of the known amount of the external standard GLC-411 (Nucheck) and C17:0-ME internal standard.

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis of Lipids

[0644] Total lipids were extracted from freeze-dried developing seeds, twelve days after flowering (daf), and mature seeds after adding a known amount of tri-C17:0-TAG as an internal quantitation standard. The extracted lipids were dissolved into 1 mL of 10 mM butylated hydroxytoluene in butanol:methanol (1:1 v/v) per 5 mg dry material and analysed using an Agilent 1200 series LC and 6410b electrospray ionisation triple quadrupole LC-MS. Lipids were chromatographically separated using an Ascentis Express RP-Amide column (50 mm×2.1 mm, 2.7 μm, Supelco) operating a binary gradient with a flow rate of 0.2 mL/min. The mobile phases were: A. 10 mM ammonium formate in H₂O:methanol:tetrahydrofuran (50:20:30 v/v/v); B. 10 mM ammonium formate in H₂O:methanol:tetrahydrofuran (5:20:75, v/v/v). Multiple reaction monitoring (MRM) lists were based on the following major fatty acids: 16:0, 18:0, 18:1, 18:2, 18:3, 18:4, 20:1, 20:2, 20:3, 20:4, 20:5, 22:4, 22:5, 22:6 using a collision energy of 30 V and fragmentor of 60 V. Individual MRM TAG was identified based on ammoniated precursor ion and product ion from neutral loss of 22:6. TAG was quantified using a 10 μM tristearin external standard.

Determination of Seed Fatty Acid Profile and Oil Content

[0645] Where seed oil content was to be determined, seeds were dried in a desiccator for 24 h and approximately 4 mg of seed was transferred to a 2 ml glass vial containing Teflon-lined screw cap. 0.05 mg triheptadecanoin dissolved in 0.1 ml toluene was added to the vial as internal standard.

[0646] Seed FAME were prepared by adding 0.7 ml of 1N methanolic HCl (Supelco) to the vial containing seed material, vortexed briefly and incubated at 80° C. for 2 h. After cooling to room temperature, 0.3 ml of 0.9% NaCl (w/v) and 0.1 ml hexane was added to the vial and mixed well for 10 min in Heidolph Vibramax 110. The FAME was collected into 0.3 ml glass insert and analysed by GC with a flame ionization detector (FID) as mentioned earlier.

[0647] The peak area of individual FAME were first corrected on the basis of the peak area responses of known amount of the same FAMEs present in a commercial standard GLC-411 (NU-CHEK PREP, INC., USA). GLC-411 contains equal amounts of 31 fatty acids (% by wt), ranging from C8:0 to C22:6. In case of fatty acids, which were not present in the standard, the inventors took the peak area responses of the most similar FAME. For example, peak area response of FAMEs of 16:1d9 was used for 16:1d7 and FAME response of C22:6 was used for C22:5. The corrected areas were used to calculate the mass of each FAME in the sample by comparison to the internal standard mass. Oil is stored mainly in the form of TAG and its weight was calculated based on FAME weight. Total moles of glycerol was determined by calculating moles of each FAMES and dividing total moles of FAMEs by three. TAG was calculated as the sum of glycerol and fatty acyl moieties using a relation: % oil by weight=100×((41× total mol FAME/3)+(total g FAME-(15× total mol FAME)))/g seed, where 41 and 15 are molecular weights of glycerol moiety and methyl group, respectively.

Analysis of the Sterol Content of Oil Samples

[0648] Samples of approximately 10 mg of oil together with an added aliquot of C24:0 monol as an internal standard were saponified using 4 mL 5% KOH in 80% MeOH and heating for 2 h at 80° C. in a Teflon-lined screw-capped glass tube. After the reaction mixture was cooled, 2 mL of Milli-Q water were added and the sterols were extracted into 2 mL of hexane:dichloromethane (4:1 v/v) by shaking and vortexing. The mixture was centrifuged and the sterol extract was removed and washed with 2 mL of Milli-Q water. The sterol extract was then removed after shaking and centrifugation. The extract was evaporated using a stream of nitrogen gas and the sterols silylated using 200 mL of BSTFA and heating for 2 h at 80° C.

[0649] For GC/GC-MS analysis of the sterols, sterol-OTMSi derivatives were dried under a stream of nitrogen gas on a heat block at 40° C. and then re-dissolved in chloroform or hexane immediately prior to GC/GC-MS analysis. The sterol-OTMS derivatives were analysed by gas chromatography (GC) using an Agilent Technologies 6890A GC (Palo Alto, Calif., USA) fitted with an Supelco Equity®-1 fused silica capillary column (15 in ×0.1 mm i.d., 0.1 μm film thickness), an FID, a split/splitless injector and an Agilent Technologies 7683B Series auto sampler and injector. Helium was the carrier gas. Samples were injected in splitless mode at an oven temperature of 120° C. After injection, the oven temperature was raised to 270° C. at 10° C. min^-1 and finally to 300° C. at 5° C. min^-1. Peaks were quantified with Agilent Technologies ChemStation software (Palo Alto, Calif., USA). GC results are subject to an error of ±5% of individual component areas.

[0650] GC-mass spectrometric (GC-MS) analyses were performed on a Finnigan Thermoquest GCQ GC-MS and a Finnigan Thermo Electron Corporation GC-MS; both systems were fitted with an on-column injector and Thermoquest Xcalibur software (Austin, Tex., USA). Each GC was fitted with a capillary column of similar polarity to that described above. Individual components were identified using mass spectral data and by comparing retention time data with those obtained for authentic and laboratory standards. A full procedural blank analysis was performed concurrent to the sample batch.

RT-PCR Conditions

[0651] Reverse transcription-PCR (RT-PCR) amplification was typically carried out using the Superscript III One-Step RT-PCR system (Invitrogen) in a volume of 25 μL using 10 pmol of the forward primer and 30 pmol of the reverse primer, MgSO₄ to a final concentration of 2.5 mM, 400 ng of total RNA with buffer and nucleotide components according to the manufacturer's instructions. Typical temperature regimes were: 1 cycle of 45° C. for 30 minutes for the reverse transcription to occur; then 1 cycle of 94° C. for 2 minutes followed by 40 cycles of 94° C. for 30 seconds, 52° C. for 30 seconds, 70° C. for 1 minute; then 1 cycle of 72° C. for 2 minutes before cooling the reaction mixtures to 5° C.

Production of B. napus Somatic Embryos by Induction with 35S-LEC2

[0652] B. napus (cv. Oscar) seeds were sterilized using chlorine gas as described by (Attila Kereszt et al., 2007). Sterilized seeds were germinated on 1/2 strength MS media (Murashige and Skoog, 1962) with 0.8% agar adjusted to pH 5.8, and grown at 24° C. under fluorescent lighting (50 μE/m² s) with a 18/6 h (light/dark) photoperiod for 6-7 days. Cotyledonary petioles with 2-4 mm stalk length were isolated aseptically from these seedlings and used as explants. Cultures of the transformed A. tumefaciens strain AGL1, one harbouring a seed specific binary vector and a second with a 35S-LEC2 construct were inoculated from single colonies from fresh plates and grown in 10 mL of LB medium with appropriate antibiotics and grown overnight at 28° C. with agitation at 150 rpm. The bacterial cells were collected by centrifugation at 4000 rpm for 5 minutes, washed with MS media containing 2% sucrose and re-suspended in 10 mL of the same medium and grown with antibiotics for selection as appropriate for 4 hours after the addition of acetosyringone to 100 μM. Two hours before addition to the plant tissues, spermidine was added to a final concentration of 1.5 mM and the final density of the bacteria adjusted to OD 600 nm=0.4 with fresh medium. The two bacterial cultures, one carrying the seed specific construct and other carrying 35S-AtLEC2, were mixed in 1:1 to 1:1.5 ratios.

[0653] Freshly-isolated B. napus cotyledonary petioles were infected with 20 mL A. tumefaciens cultures for 6 minutes. The cotyledonary petioles were blotted on sterile filter paper to remove excess A. tumefaciens and then transferred to co-cultivation media (MS media with 1 mg/L TDZ, 0.1 mg/L NAA, 100 μM acetosyringone supplemented with L-cysteine (50 mg/L), ascorbic acid (15 mg/L) and MES (250 mg/l)). The plates were sealed with micro-pore tape and incubated in the dark at 24° C. for 48 hrs. The co-cultivated explants were transferred to pre-selection media (MS containing 1 mg/L TDZ, 0.1 mg/L NAA, 3 mg/L AgNO₃, 250 mg/L cefotaxime and 50 mg/L timentin) and cultured for 4-5 days at 24° C. with a 16 h/8 h photoperiod. The explants were then transferred to selection media (MS containing 1 mg/L TDZ, 0.1 mg/L NAA, 3 mg/L AgNO₃, 250 mg/L cefotaxime and 50 mg/L timentin) according to the selectable marker gene on the seed specific vector and cultured for 2-3 weeks at 24° C. with a 16 h/8 h photoperiod. Explants with green embryogenic callus were transferred to hormone free MS media (MS with 3 mg/L AgNO₃, 250 mg/L cefotaxime, 50 mg/L timentin and the selection agent) and cultured for another 2-3 weeks. Torpedo or cotyledonary stage embryos isolated from surviving explants on the selection medium were analysed for fatty acid composition in their total lipid using GC.

Example 2

Stable Expression of Transgenic DHA Pathways in Arabidopsis thaliana Seeds

Binary Vector Construction

[0654] The binary vectors pJP3416-GA7 and pJP3404 each contained seven heterologous fatty acid biosynthesis genes, encoding 5 desaturases and 2 elongases, and a plant selectable marker between the left and right border repeats of the T-DNA present in each vector (FIGS. 2 and 3). SEQ ID NO:1 provides the nucleotide sequence of the T-DNA region of pJP3416-GA7 from the right to left border sequences. Both genetic constructs contained plant codon-optimised genes encoding a Lachancea kluyveri Δ12-desaturase (comprising nucleotides 14143-16648 of SEQ ID NO:1), a Pichia pastoris ω3-desaturase (comprising nucleotides 7654-10156 of SEQ ID NO: 1), a Micromonas pusilla Δ6-desaturase (comprising nucleotides 226-2309 of SEQ ID NO: 1), Pavlova salina Δ5- and Δ4-desaturases (comprising nucleotides 4524-6485 and 10157-14142 of SEQ ID NO:1, respectively) and Pyramimonas cordata Δ6- and Δ5-elongases (comprising nucleotides 2310-4523 and 17825-19967 of SEQ ID NO:1, respectively). The specific regions of the T-DNA (Orientation: right to left border sequences) region of the binary vector pJP3416-GA7 with respect to SEQ ID NO:1 are as follows:

Nucleotides 1-163: Right border; 480-226, Agrobacterium tumefaciens nopaline synthase terminator (TER_NOS); 1883-489, Micromonas pusilla Δ6-desaturase; 2309-1952, Brassica napus truncated napin promoter (PRO_FP1); 2310-3243, Arabidopsis thaliana FAE1 promoter (PRO_FAE1); 3312-4181, Pyramimonas cordata Δ6-elongase; 4190-4523, Glycine max lectin terminator (TER_Lectin); 4524-4881, PRO_FP; 4950-6230: Pavlova salina Δ5-desaturase; 6231-6485: TER_NOS; 7653-6486, Nicotiana tabacum Rb7 matrix attachment region (MAR); 8387-7654, Linum usitatissimum conlinin1 terminator (TER_Cnl1); 9638-8388, Pichia pastoris ω3-desaturase; 10156-9707, Linum usitatissimum conlinin1 promoter (PRO_Cnl1); 10157-12189, Linum usitatissimum conlinin1 promoter; 12258-13604, Pavlova salina Δ4-desaturase; 13605-14142, Linum usitatissimum conlinin2 terminator; 14143-14592, PRO_Cnl1; 14661-15914, Lachancea kluyveri Δ12-desaturase; 15915-16648, TER_Cnl1; 17816-16649, MAR; 17825-18758, PRO_FAE1; 18827-19633, Pyramimonas cordata Δ5-elongase; 19634-19967, TER_Lectin; 19990-20527, Cauliflower mosaic virus 35S promoter with duplicated enhancer region; 20537-21088, Streptomyces viridochromogenes phosphinothricin-N-acetyltransferase; 21097-21349, TER_NOS; 21367-21527, Left border.

[0655] The seven coding regions in the constructs were each under the control of a seed specific promoter--three different promoters were used, namely the truncated Brassica napus napin promoter (pBnFP1), the Arabidopsis thaliana FAE1 promoter (pAtFAE1) and the Linum usitatissimum conlinin 1 promoter (pLuCnl1). The seven fatty acid biosynthesis genes together coded for an entire DHA synthesis pathway that was designed to convert 18:1.sup.Δ9 (oleic acid) through to 22:6.sup.Δ4, 7, 10, 13, 19 (DHA). Both binary vectors contained a BAR plant selectable marker coding region operably linked to a Cauliflower Mosaic Virus (CaMV) 35S promoter with duplicated enhancer region and A. tumefaciens nos3' polyadenylation region-transcription terminator. The plant selectable marker was situated adjacent to the left border of the T-DNA region, therefore distally located on the T-DNA with respect to the orientation of T-DNA transfer into the plant cells. This increased the likelihood that partial transfer of the T-DNA, which would be likely to not include the selectable marker gene, would not be selected. pJP3416-GA7 and pJP3404 each contained an RiA4 origin of replication from Agrobacterium rhizogenes (Hamilton, 1997).

[0656] pJP3416-GA7 was generated by synthesising the DNA region corresponding to nucleotides 226-19975 of SEQ ID NO: 1 (GA7 region) and inserting this region into the recipient binary vector pJP3416 at the PspOMI site. Each fatty acid biosynthetic gene on GA7 included a Tobacco Mosaic Virus 5' untranslated region (5'UTR) sequence which was operably linked to each coding region, between the promoter and the translation initiation ATG, to maximise translation efficiency of the mRNAs produced from the genes. The GA7 construct also included two Nicotiana tabacum Rb7 matrix attachment region (MAR) sequences, as described by Hall et al. (1991). MAR sequences, sometimes termed nuclear attachment regions, are known to bind specifically to the nuclear matrix in vitro and may mediate binding of chromatin to the nuclear matrix in vivo. MARs are thought to function to reduce transgene silencing. In pJP3416-GA7 the MARs were also inserted and positioned within the T-DNA region in order to act as DNA spacers to insulate transgenic expression cassettes. The pJP3416 vector prior to insertion of the GA7 region contained only the plant selectable marker cassette between the borders.

[0657] The genetic construct pJP3404 was made by sequential restriction enzyme-based insertions in which gene cassettes were added to the binary vector, pJP3367, which comprised genes for production of SDA in seeds. This construct contained genes encoding the L. kluyveri Δ12-desaturase and P. pastoris ω3-desaturase, both expressed by the B. napus truncated napin promoter (FP1), and the M. pusilla Δ6-desaturase expressed by the A. thaliana FAE1 promoter (FIG. 4). First, the A. thaliana FAD2 intron was flanked by EcoRI sites and cloned into the pJP3367 MfeI site to generate pJP3395. A fragment containing the P. cordata Δ6- and Δ5-elongase cassettes driven by the FAE1 and FP1 promoters, respectively, was cloned into the KasI site of pJP3395 to generate pJP3398. pJP3399 was then generated by replacing the RK2 origin of replication in pJP3398 with a RiA4 origin of replication. The final binary vector, pJP3404, was generated by cloning a SbfI-flanked fragment containing the P. salina Δ5- and Δ4-desaturase cassettes driven by the FP1 and FAE1 promoters, respectively, into the SbfI site of pJP3399.

A. thaliana Transformation and Analysis of Fatty Acid Composition

[0658] The chimeric vectors were introduced into A. tumefaciens strain AGL1 and cells from cultures of the transformed Agrobacterium used to treat A. thaliana (ecotypes Columbia and a fad2 mutant) plants using the floral dip method for transformation (Clough and Bent, 1998). After maturation, the T₁ seeds from the treated plants were harvested and plated onto MS plates containing PPT for selection of plants containing the BAR selectable marker gene. Surviving, healthy T₁ seedlings were transferred to soil. After growth of the plants to maturity and allowing for self-fertilisation, T₂ seeds from these plants were harvested and the fatty acid composition of their seed lipid analysed by GC analysis as described in Example 1.

[0659] The data for the DHA level in the seed lipids are shown in FIG. 5 (lanes labelled T₂) for 13 transformants using pJP3416-GA7 into the Columbia genetic background, and for six transformants using the fad2 mutant. The pJP3416-GA7 construct resulted in the production of slightly higher levels of DHA, as a percentage of total fatty acid content, on average than the pJP3404 construct. Table 4 shows the fatty acid composition of total seed lipid from the T₂ lines with the highest DHA levels. The calculated conversion efficiencies for each enzymatic step in the production of DHA from oleic acid in the same seeds are shown in Table 5. Conversion efficiencies were calculated as (% products×100)/(% remaining substrate+% products), thereby expressed as a percentage.

[0660] The highest observed level of DHA produced in the pJP3416-GA7 T₂ transformed lines was 6.2%, additionally with 0.5% EPA and 0.2% DPA (line #14). These T₂ seeds were still segregating for the transgene i.e. were not yet uniformly homozygous. Compiled data from the total seed lipid profiles from independent transgenic seed (Table 4) are shown in Table 6. The level of ω3 fatty acids produced as a result of the transgenes in these seeds (total new ω3 fatty acids, excluding the level of ALA which was produced endogenously in the Columbia background) was 10.7% while the level of ω6 fatty acids (total new ω36 fatty acids but excluding 18:2.sup.Δ9, 12) was 1.5%. This represents an extremely favourable ration of new ω3 fatty acids:new ω6 fatty acids, namely 7.3:1.

[0661] T₂ seeds of selected lines transformed with pJP3416-GA7, namely for lines designated 7, 10, 14, 22 and 34 in the Columbia background and for lines designated 18, 21 and 25 in the fad2 mutant background, were plated onto MS media containing PPT for selection of transgenic seedlings in vitro. Twenty PPT-resistant seedlings for each line were transferred to soil and grown to maturity after self-fertilisation. These plants were highly likely to be homozygous for the selectable marker gene, and therefore for at least one T-DNA insertion in the genome of the plants. T₃ seed from these plants were harvested and analysed for fatty acid composition in their seedoil by GC. The data are shown in Table 7. This analysis revealed that the pJP3416-GA7 construct generated higher levels of the ω3 LC-PUFA DHA in T₃ seeds of the homozygous plants than in the segregating T₂ seed. Up to about 13.9% DHA was observed in the T₃ pJP3416-GA7 transformed line designated 22.2 in the Columbia background, increased from about 5.5% in the hemizygous T₂ seed, with a sum level of about 24.3% of new ω3 fatty acids as a percentage of the total fatty acids in the seed lipid content. New ω6 fatty acids were at a level of 1.1% of total fatty acids, representing a very favourable ratio of new ω3 fatty acids:new ω6 fatty acids, namely about 22:1. Similarly, transformants in the fad2 mutant background yielded 20.6% as a sum of new ω3 fatty acids, including 11.5% DHA, as a percentage of the total fatty acids in the seed lipid content.

TABLE-US-00004 TABLE 4 Fatty acid composition of total seed lipid from independent transgenic T₂ Arabidopsis seeds with DHA levels at the higher end of the observed range. `Col` refers to the Columbia ecotype and `FAD2` to the fad2 mutant ecotype. `GA7` refers to transformation with the T-DNA of the pJP3416-GA7 vector, pJP3404 with the T- DNA of the pJP3404 vector. 20:1n-9 and 20:1n-11 fatty acids were not resolved in the GC analysis. "Other minor" fatty acids include 14:0, 16:1n7, 16:1n9, 16:1n13t, 16:2n6, 16:3n3, i18:0, 18:1n5, 20:1n5, 22:0, 22:1n7, 22:1n11/n13, 24:0, 24:1n9. pJP3404_Col_#1 pJP3404_FAD2_#31 GA7_Col_#7 GA7_Col_#34 GA7_Col_#2 GA7_Col_#10 16:0 9.6 7.8 8.7 8.2 8.7 8.6 18:0 2.9 3.9 3.7 3.9 3.6 3.3 18:1d11 2.2 1.8 2.0 1.9 2.0 2.3 20:0 1.6 2.3 2.0 2.0 2.1 1.6 20:1d13 2.2 1.8 1.6 1.5 1.7 1.6 20:1d9/d11 13.0 15.9 16.1 16.1 16.3 15.0 22:1d13 1.1 1.2 1.1 1.1 1.3 1.0 Other 1.9 1.5 1.5 1.4 1.5 1.3 minor 18:1d9 10.8 14.0 10.6 10.6 10.1 11.1 18:2ω6 28.9 28.3 16.4 16.1 18.2 13.7 18:3ω3 16.6 14.9 29.6 29.6 27.5 32.4 18:3ω6 0.7 0.5 0.1 0.1 0.1 0.1 20:2ω6 1.6 1.5 1.1 1.2 1.3 1.0 20:3ω6 0.0 0.0 0.0 0.0 0.0 0.0 20:4ω6 0.0 0.0 0.0 0.0 0.0 0.0 22:4ω6 1.6 0.6 0.3 0.3 0.3 0.4 22:5ω6 0.1 0.1 0.0 0.0 0.0 0.0 18:4ω3 1.0 0.5 1.2 1.1 1.1 1.5 20:3ω3 0.0 0.0 0.0 0.6 0.0 0.0 20:4ω3 0.4 0.6 0.6 0.7 0.5 0.8 20:5ω3 0.2 0.2 0.3 0.3 0.3 0.3 22:5ω3 0.0 0.2 0.2 0.2 0.2 0.2 22:6ω3 3.6 2.4 3.0 3.1 3.3 3.9 GA7_Col_#22 GA7_Col_#14 GA7_FAD2_#25 GA7_FAD2_#21 GA7_FAD2_#18 16:0 8.3 9.7 7.2 8.5 7.5 18:0 3.4 3.6 3.2 3.9 3.0 18:1d11 2.3 2.7 1.9 2.0 1.8 20:0 1.6 1.8 1.6 2.2 1.5 20:1d13 1.5 1.7 1.5 1.7 1.4 20:1d9/d11 13.9 13.5 18.3 15.9 17.0 22:1d13 1.0 1.0 1.0 1.3 1.2 Other 1.6 1.7 1.6 1.4 1.6 minor 18:1d9 10.0 7.7 26.0 8.2 20.9 18:2ω6 13.7 11.4 6.6 16.6 4.3 18:3ω3 30.4 32.8 21.9 27.7 30.1 18:3ω6 0.2 0.1 0.1 0.2 0.1 20:2ω6 1.0 1.0 0.4 1.4 0.4 20:3ω6 0.0 0.0 0.0 0.0 0.0 20:4ω6 0.0 0.0 0.0 0.0 0.0 22:4ω6 0.5 0.4 0.5 0.4 0.4 22:5ω6 0.0 0.0 0.0 0.0 0.0 18:4ω3 2.7 2.7 1.9 1.8 1.7 20:3ω3 0.6 0.7 0.0 0.8 0.6 20:4ω3 0.8 0.4 1.0 0.8 0.8 20:5ω3 0.7 0.5 0.6 0.4 0.5 22:5ω3 0.2 0.2 0.3 0.3 0.3 22:6ω3 5.5 6.2 4.3 4.4 4.8

TABLE-US-00005 TABLE 5 Conversion efficiencies of the individual enzymatic steps for production of DHA from oleic acid, observed in total seed lipid from independent transgenic seed as for Table 4. pJP3404_Col_#1 pJP3404_FAD2_#31 GA7_Col_#7 GA7_Col_#34 GA7_Col_#2 GA7_Col_#10 d12-des 69.6% 62.5% 66.4% 66.6% 66.7% 67.5% d15-des 39.8% 37.8% 66.1% 66.8% 62.3% 72.1% Omega-6 d6-des 4.5% 2.5% 0.7% 0.7% 0.7% 0.9% (d9-elo) 3.1% 3.1% 2.2% 2.3% 2.4% 1.8% d6-elo 71.4% 56.9% 83.3% 83.4% 83.0% 84.7% d5-des 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% d5-elo 100.0% 97.8% 100.0% 100.0% 100.0% 100.0% d4-des 6.2% 13.0% 0.0% 0.0% 0.0% 0.0% Omega-3 d6-des 23.9% 21.0% 15.2% 15.4% 16.4% 17.1% (d9-elo) 0.0% 0.0% 0.0% 1.8% 0.0% 0.0% d6-elo 80.6% 86.6% 77.7% 79.6% 79.4% 77.5% d5-des 93.7% 92.1% 91.7% 91.4% 91.5% 92.6% d5-elo 93.7% 92.1% 91.7% 91.4% 91.5% 92.6% d4-des 100.0% 90.6% 94.8% 94.0% 95.3% 94.4% GA7_Col_#22 GA7_Col_#14 GA7_FAD2_#25 GA7_FAD2_#21 GA7_FAD2_#18 d12-des 70.2% 72.7% 45.9% 69.5% 53.7% d15-des 72.7% 77.2% 79.7% 66.0% 88.1% Omega-6 d6-des 1.3% 1.0% 1.6% 1.1% 1.1% (d9-elo) 1.8% 1.7% 1.2% 2.7% 0.9% d6-elo 70.3% 74.5% 85.5% 66.1% 88.0% d5-des 100.0% 100.0% 100.0% 100.0% 100.0% d5-elo 100.0% 100.0% 100.0% 100.0% 100.0% d4-des 0.0% 0.0% 0.0% 0.0% 0.0% Omega-3 d6-des 24.7% 23.6% 27.1% 21.9% 21.0% (d9-elo) 2.0% 2.2% 0.0% 2.6% 2.1% d6-elo 72.7% 73.0% 76.7% 77.4% 79.2% d5-des 89.6% 92.4% 88.0% 91.8% 91.0% d5-elo 89.6% 92.4% 88.0% 91.8% 91.0% d4-des 95.8% 96.9% 93.1% 92.9% 94.2%

TABLE-US-00006 TABLE 6 Compiled data from the total seed lipid profiles from independent transgenic seed shown in Table 2. Calculations do not include the `minor fatty acids` in Table 4. Parameter pJP3404_Col_#1 pJP3404_FAD2_#31 GA7_Col_#7 GA7_Col_#34 GA7_Col_#2 GA7_Col_#10 total w3 (% of total FA) 21.8 18.8 34.9 35.6 32.9 39.1 total w6 (% of total FA) 32.9 31.0 17.9 17.7 19.9 15.2 w3/w6 ratio 0.66 0.61 1.95 2.01 1.65 2.57 w6/w3 ratio 1.51 1.65 0.51 0.50 0.60 0.39 total novel w3 (% of total FA) 5.2 3.9 5.3 6.0 5.4 6.7 total novel w6 (% of total FA) 4.0 2.7 1.5 1.6 1.7 1.5 novel w3/w6 ratio 1.30 1.44 3.53 3.75 3.18 4.47 novel w6/w3 ratio 0.77 0.69 0.28 0.27 0.31 0.22 OA to EPA efficiency 4.8% 3.5% 4.3% 4.4% 4.7% 5.4% OA to DHA efficiency 4.5% 3.0% 3.7% 3.8% 4.1% 4.8% LA to EPA efficiency 6.9% 5.6% 6.6% 6.8% 7.2% 8.1% LA to DHA efficiency 6.6% 4.8% 5.7% 5.8% 6.3% 7.2% ALA to EPA efficiency 17.4% 14.9% 10.0% 10.1% 11.6% 11.3% ALA to DHA efficiency 16.5% 12.8% 8.6% 8.7% 10.0% 10.0% total saturates 14.1 14.0 14.4 14.1 14.4 13.5 total monounsaturates 29.3 34.7 31.4 31.2 31.4 31.0 total polyunsaturates 54.7 49.8 52.8 53.3 52.8 54.3 total C20 17.4 20 19.7 20.4 20.1 18.7 total C22 6.4 4.5 4.6 4.7 5.1 5.5 C20/C22 ratio 2.72 4.44 4.28 4.34 3.94 3.40 Parameter GA7_Col_#22 GA7_Col_#14 GA7_FAD2_#25 GA7_FAD2_#21 GA7_FAD2_#18 total w3 (% of total FA) 40.9 43.5 30.0 36.2 38.8 total w6 (% of total FA) 15.4 12.9 7.6 18.6 5.2 w3/w6 ratio 2.66 3.37 3.95 1.95 7.46 w6/w3 ratio 0.38 0.30 0.25 0.51 0.13 total novel w3 (% of total FA) 10.5 10.7 8.1 8.5 8.7 total novel w6 (% of total FA) 1.7 1.5 1.0 2.0 0.9 novel w3/w6 ratio 6.18 7.13 8.10 4.25 9.67 novel w6/w3 ratio 0.16 0.14 0.12 0.24 0.10 OA to EPA efficiency 7.9% 8.8% 6.3% 6.4% 6.7% OA to DHA efficiency 6.8% 7.9% 5.2% 5.5% 5.8% LA to EPA efficiency 11.4% 12.2% 13.8% 9.3% 12.7% LA to DHA efficiency 9.8% 11.0% 11.4% 8.0% 10.9% ALA to EPA efficiency 15.6% 15.9% 17.3% 14.1% 14.4% ALA to DHA efficiency 13.4% 14.3% 14.3% 12.2% 12.4% total saturates 13.3 15.1 12.0 14.6 12.0 total monounsaturates 28.7 26.6 48.7 29.1 42.3 total polyunsaturates 56.3 56.4 37.6 54.8 44.0 total C20 18.5 17.8 21.8 21 20.7 total C22 7.2 7.8 6.1 6.4 6.7 C20/C22 ratio 2.57 2.28 3.57 3.28 3.09

TABLE-US-00007 TABLE 7 Fatty acid composition of total seed lipid from independent transgenic T₃ and T₄ Arabidopsis progeny seeds obtained from plant lines as in Table 3. The error shown in the T₄ generation denotes the SD of n = 10. GA7_Col_7.2 GA7_Col_34.2 GA7_Col_10.13 GA7_Col_22.2 GA7_Col_14.19 16:0 9.8 9.0 9.5 11.2 10.4 18:0 4.0 3.8 4.2 3.4 3.5 18:1n7 2.0 1.9 2.2 2.9 2.5 20:0 2.2 1.9 1.7 1.4 2.3 20:1d13 1.4 1.3 1.2 1.6 2.5 20:1d9/11 13.6 14.7 12.4 9.5 13.0 22:1d13 1.2 1.2 0.8 0.6 1.6 Other 1.8 1.5 1.5 2.1 2.6 minor 18:1d9 5.5 6.7 6.8 4.6 6.9 18:2ω6 7.5 7.9 7.4 5.6 14.8 18:3ω3 33.7 33.7 36.1 31.5 26.1 18:3ω6 0.2 0.2 0.2 0.4 0.1 20:2ω6 1.0 1.0 0.7 0.7 1.4 20:3ω6 0 0 0 0 0 20:4ω6 0 0 0 0 0 22:4ω6 0 0 0 0 0 22:5ω6 0 0 0 0 0 18:4ω3 3.1 2.6 3.0 5.3 3.3 20:3ω3 1.4 1.3 1.2 1.3 1.2 20:4ω3 0.7 0.6 0.6 0.9 0.2 20:5ω3 0.9 0.9 0.7 1.9 0.8 22:5ω3 0.7 0.6 0.6 1.0 0.4 22:6ω3 9.5 9.2 9.4 13.9 6.6 GA7_FAD2- GA7_FAD2- GA7_FAD2- T₄ Col_22.2 T₄ Col_22.2 25.10 (mean ± SD) best line 16:0 8.1 10.7 7.7 10.6 ± 0.9 12.2 18:0 3.5 3.8 3.3 3.5 ± 0.4 3.6 18:1n7 1.7 2.2 1.6 2.3 ± 0.2 2.6 20:0 1.8 2.0 1.9 1.9 ± 0.3 2.0 20:1d13 1.2 1.4 1.3 1.6 ± 0.2 1.9 20:1d9/11 15.7 12.4 18.4 11.7 ± 1.7 9.5 22:1d13 1.0 1.1 1.5 0.9 ± 0.1 0.8 Other 1.7 1.9 1.6 1.9 ± 0.1 2.3 minor 18:1d9 11.3 4.2 11.5 4.6 ± 1.0 3.3 18:2ω6 5.8 8.9 5.6 5.3 ± 0.9 4.3 18:3ω3 28.3 28.9 30.8 31.0 ± 1.1 29.5 18:3ω6 0.3 0.6 0.1 0.4 ± 0.1 0.4 20:2ω6 0.6 1.2 0.6 0.9 ± 0.1 0.9 20:3ω6 0 0 0 20:4ω6 0 0 0 22:4ω6 0 0 0 0.1 ± 0.0 0.1 22:5ω6 0 0 0 18:4ω3 3.7 5.2 2.6 4.8 ± 0.9 5.5 20:3ω3 1.1 1.3 1.3 1.5 ± 0.2 1.7 20:4ω3 1.7 0.9 0.9 0.8 ± 0.2 0.8 20:5ω3 1.2 1.0 0.8 1.5 ± 0.3 1.8 22:5ω3 0.8 0.6 0.5 1.1 ± 0.2 1.5 22:6ω3 10.3 11.5 7.9 13.3 ± 1.6 15.1 indicates data missing or illegible when filed

[0662] Enzymatic conversion efficiencies for each enzyme step in the pathway for production of DHA from oleic acid are shown in Table 8 for the T₃ seeds with the higher DHA levels. The Δ12-desaturase conversion efficiency in seeds of line 22.2 was 81.6% and the ω3-desaturase efficiency was 89.1%, both of them remarkably high and indicating that these fungal (yeast) enzymes were able to function well in developing seeds. The activities of the other exogenous enzymes in the DHA pathway were similarly high for ω3 substrates with the Δ6-desaturase acting at 42.2% efficiency, Δ6-elongase at 76.8%, Δ5-desaturase at 95.0%, Δ5-elongase at 88.7% and Δ4-desaturase at 93.3% efficiency. The Δ6-desaturase activity on the ω6 substrate LA was much lower, with the Δ6-desaturase acting at only 0.7% conversion efficiency on LA. GLA was present at a level of only 0.4% and was the only new ω6 product aside from 20:2ω6 detected in the T₃ seeds with the highest DHA content. Compiled data from the total seed lipid profiles from independent transgenic seed (Table 7) are shown in Table 9. This data for the line with the greatest DHA level included a total ω6 FA (including LA) to total ω3 FA (including ALA) ratio of 0.10. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA) ratio in the lipid of this line was 0.05. Total polyunsaturated fatty acid levels were more than 50% in these lines, and greater than 60% in at least 4 of the lines. Overall conversion efficiencies were calculated to be: OA to EPA=21.8%, OA to DHA=18.0%, LA to EPA=26.9%, LA to DHA=22.2%, ALA to EPA=30.1%, ALA to DHA=24.9%.

TABLE-US-00008 TABLE 8 Conversion efficiencies of the individual enzymatic steps for the production of DHA from oleic acid, observed in total seed lipid from transgenic T₃ Arabidopsis seeds as in Table 7. GA7_Col_7.2 GA7_Col_34.2 GA7_Col_10.13 GA7_Col_22.2 GA7_Col_14.19 d12-des 75.4% 73.1% 75.7% 81.6% 73.4% d15-des 85.3% 84.4% 86.2% 89.1% 70.2% Omega-6 d6-des 0.3% 0.3% 0.3% 0.7% 0.3% (d9-elo) 1.7% 1.7% 1.2% 1.2% 2.6% d6-elo d5-des d5-elo d4-des Omega-3 d6-des 30.7% 29.3% 28.2% 42.2% 30.2% (d9-elo) 2.7% 2.7% 2.3% 2.4% 3.0% d6-elo 79.0% 81.1% 79.0% 76.8% 70.9% d5-des 94.0% 94.6% 94.5% 95.0% 97.9% d5-elo 91.9% 91.7% 93.6% 88.7% 89.5% d4-des 93.2% 93.7% 94.4% 93.3% 93.7% GA7_FAD2- GA7_FAD2- T₄ Col_22.2 T₄ Col_22.2 25.10 GA7_FAD2-21.2 18.14 (mean) best line d12-des 66.6% 78.5% 63.1% 67.6% 82.7% d15-des 87.5% 82.2% 87.6% 81.0% 90.9% Omega-6 d6-des 0.6% 1.0% 0.2% 1.3% 0.7% (d9-elo) 1.1% 2.0% 1.3% 1.6% 1.5% d6-elo d5-des d5-elo d4-des Omega-3 d6-des 38.5% 40.0% 29.2% 41.0% 45.7% (d9-elo) 2.3% 2.7% 2.9% 2.8% 3.1% d6-elo 79.2% 73.2% 79.1% 77.5% 77.7% d5-des 87.8% 93.3% 91.1% 95.0% 95.8% d5-elo 89.9% 92.2% 91.6% 90.8% 90.2% d4-des 92.5% 95.0% 93.9% 92.2% 90.9%

TABLE-US-00009 TABLE 9 Compiled data from the total seed lipid profiles from independent transgenic seed shown in Table 5. Calculations do not include the `minor fatty acids` in Table 7. Parameter GA7-Col_7.2 GA7-Col_34.2 GA7-Col_10.13 GA7-Col_22.2 GA7-Col_14.19 total w3 (% of total FA) 50.0 48.9 51.6 55.8 38.6 total w6 (% of total FA) 8.7 9.1 8.3 6.7 16.3 w3/w6 ratio 5.75 5.37 6.22 8.33 2.37 w6/w3 ratio 0.17 0.19 0.16 0.12 0.42 total novel w3 (% of total FA) 16.3 15.2 15.5 24.3 12.5 total novel w6 (% of total FA) 1.2 1.2 0.9 1.1 1.5 novel w3/w6 ratio 13.58 12.67 17.22 22.09 8.33 novel w6/w3 ratio 0.07 0.08 0.06 0.05 0.12 OA to EPA efficiency 14.1% 13.3% 13.4% 21.8% 10.2% OA to DHA efficiency 12.0% 11.4% 11.8% 18.0% 8.6% LA to EPA efficiency 18.9% 18.4% 17.9% 26.9% 14.2% LA to DHA efficiency 16.2% 15.9% 15.7% 22.2% 12.0% ALA to EPA efficiency 22.2% 21.9% 20.7% 30.1% 20.2% ALA to DHA efficiency 19.0% 18.8% 18.2% 24.9% 17.1% total saturates 16.0 14.7 15.4 16.0 16.2 total monounsaturates 23.7 25.8 23.4 19.2 26.5 total polyunsaturates 58.7 58.0 59.9 62.5 54.9 total C20 19 19.8 16.8 15.9 19.1 total C22 11.4 11 10.8 15.5 8.6 C20/C22 ratio 1.67 1.80 1.56 1.03 2.22 T₄ Col_22.2 T₄ Col_22.2 Parameter GA7-FAD2-25.10 GA7-FAD2-21.2 GA7-FAD2-18.14 (mean ± SD) best line total w3 (% of total FA) 47.1 49.4 44.8 54.0 55.9 total w6 (% of total FA) 6.7 10.7 6.3 6.7 5.7 w3/w6 ratio 7.03 4.62 7.11 8.06 9.81 w6/w3 ratio 0.14 0.22 0.14 0.12 0.10 total novel w3 (% of total FA) 18.8 20.5 14.0 23.0 26.4 total novel w6 (% of total FA) 0.9 1.8 0.7 1.4 1.4 novel w3/w6 ratio 20.89 11.39 20.00 16.43 18.86 novel w6/w3 ratio 0.05 0.09 0.05 0.06 0.05 OA to EPA efficiency 15.0% 16.8% 11.2% 20.4% 24.5% OA to DHA efficiency 12.6% 14.8% 9.6% 17.1% 20.1% LA to EPA efficiency 22.9% 21.8% 18.0% 26.2% 29.9% LA to DHA efficiency 19.1% 19.1% 15.5% 21.9% 24.5% ALA to EPA efficiency 26.1% 26.5% 20.5% 29.4% 32.9% ALA to DHA efficiency 21.9% 23.3% 17.6% 24.6% 27.0% total saturates 13.4 16.5 12.9 16.0 17.8 total monounsaturates 30.9 21.3 34.3 21.1 18.1 total polyunsaturates 53.8 60.1 51.1 60.7 61.6 total C20 21.5 18.2 23.3 18 16.6 total C22 12.1 13.2 9.9 15.4 17.5 C20/C22 ratio 1.78 1.38 2.35 1.17 0.95

[0663] T₃ seeds from the pJP3416-GA7 line 22.2 in the Columbia background, which were progeny from T₂ line 22, were sown directly to soil and the fatty acid composition of mature seed from the resultant T₃ plants analysed by GC. The average DHA level of these seeds was 13.3%±1.6 (n=10) as a percentage of total fatty acids in the seed lipid. As shown in Table 6 (right hand column), the line with the highest level of DHA contained 15.1% DHA in the total fatty acids of the seed lipid. The enzymatic conversion efficiencies are shown in Table 8 for each step in the production of DHA from oleic acid.

[0664] The total ω6 FA (including LA) to ω3 FA (including ALA) ratio in the line with the highest DHA level was 0.102. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA) ratio in the line with the highest DHA level was 0.053. The level of total saturated fatty acids was about 17.8% and the level of monounsaturated fatty acids was about 18.1%. The level of total ω6-fatty acids was about 5.7% and the level of ω3-fatty acids was about 55.9%. Overall conversion efficiencies were calculated to be: OA to EPA=24.5%, OA to DHA=20.1%, LA to EPA=29.9%, LA to DHA=24.5%, ALA to EPA=32.9%, ALA to DHA=27.0%. Total omega-3 fatty acids were found to accumulate to 55.9% of total fatty acids whereas omega-6 fatty acids were 5.7% of the total profile.

[0665] Southern blot hybridisation analysis was performed. The results showed that the high-accumulating DHA lines were either single- or double-copy for the T-DNA from the pJP3416-GA7 construct with the exception of transgenic line Columbia #22, which had three T-DNA insertions in the genome of the Arabidopsis plant. The T5 generation seed was also analysed and found to have up to 13.6% DHA in the total seed lipids. The GA7 construct was found to be stable across multiple generations in terms of DHA production capability.

Determination of Oil Content in Transgenic A. thaliana DHA Lines

[0666] The oil content of transgenic A. thaliana seeds with various levels of DHA was determined by GC as described in Example 1. The data are shown in FIG. 6, graphing the oil content (% oil by weight of seed) against the DHA content (as a percentage of total fatty acids). Up to 26.5 mg of DHA per gram of seed was observed (Table 10). The oil content of the transgenic Arabidopsis seeds was found to be negatively correlated with DHA content. The amount of DHA per weight of seed was greater in the transformed seeds with a DHA level of about 9% relative to the seeds with about 14% DHA. Whether this would be true for seeds other than Arabidopsis has not been determined.

TABLE-US-00010 TABLE 10 Proportion and amount of DHA in GA7-transformed Arabidopsis seeds. DHA content DHA content Oil content per weight (% of TFA) (% oil per g seeds) (mg/g seed) GA7/col 22.2-1 14.2 14.89 20.2 GA7/col 22.2-2 14.3 15.02 20.5 GA7/col 22.2-3 14.0 15.92 21.2 GA7/col 10.15-1 8.7 30.23 25.06 GA7/col 10.15-2 8.6 31.25 25.77 GA7/col 10.15-3 8.8 31.70 26.49

Example 3

Stable Expression of a Transgenic DHA Pathway in Camelina sativa Seeds

[0667] The binary vector pJP3416-GA7 as described above was introduced into A. tumefaciens strain AGL1 and cells from a culture of the transformed Agrobacterium used to treat C. sativa flowering plants using a floral dip method for transformation (Lu and Kang, 2008). After growth and maturation of the plants, the T₁ seeds from the treated plants were harvested, sown onto soil and the resultant plants treated by spraying with the herbicide BASTA to select for plants which were transgenic for, and expressing, the bar selectable marker gene present on the T-DNA of pJP3416-GA7. Surviving T₁ plants which were tolerant to the herbicide were grown to maturity after allowing them to self-fertilise, and the resultant T₂ seed harvested. Five transgenic plants were obtained, only three of which contained the entire T-DNA.

[0668] Lipid was extracted from a pool of approximately twenty seeds from each of the three plants that contained the entire T-DNA. Two of the pooled samples contained very low, barely detectable levels of DHA, but the third pool contained about 4.7% DHA (Table 12). Therefore, lipid was extracted from 10 individual T₂ seeds from this plant and the fatty acid composition analysed by GC. The fatty acid composition data of the individual seeds for this transformed line is also shown in Table 1. Compiled data from the total seed lipid profiles (Table 11) are shown in Table 12.

TABLE-US-00011 TABLE 11 Fatty acid composition of total seed lipids from transgenic T₂ Camelina sativa seeds transformed with the T-DNA from pJP3416-GA7. The fatty acid composition is shown for a pooled seed batch (FD5.46) and for 10 single seeds ranked (left to right) from highest to lowest DHA. FD5.46 Fatty acid pooled # 2 # 4 # 8 # 7 # 9 # 1 # 3 # 5 # 6 # 10 14:0 0 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.1 0.2 0.2 16:0 11.6 12.1 12.3 12.1 13.2 12.3 12.8 11.9 11.4 11.5 11.7 16:1 0.2 0.0 0.1 0.1 0.0 0.2 0.0 0.2 0.2 0.2 0.2 16:3 0.3 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 18:0 3.7 3.3 3.2 3.2 3.0 3.1 3.2 3.3 3.1 3.2 3.2 18:1 10.8 8.0 8.0 8.6 8.5 9.4 11.0 10.2 8.3 9.4 8.6 18:1d11 1.7 1.3 1.4 1.4 1.7 1.4 1.5 1.3 1.3 1.3 1.3 18:2 24.7 18.2 19.5 19.2 18.5 20.1 23.8 32.2 30.3 29.8 31.6 18:3ω3 27.4 26.7 26.6 27.3 28.9 28.2 27.4 28.3 29.2 29.5 28.2 18:3ω6 0.2 1.4 0.3 0.3 0.4 0.2 0.5 0.0 0.5 0.4 0.6 20:0 1.6 1.4 1.3 1.4 1.2 1.4 1.4 1.8 2.1 1.9 2.0 18:4ω3 2.2 6.8 6.4 5.7 7.2 5.7 4.1 0.0 0.0 0.0 0.0 20:1d11 5.3 4.4 4.6 4.8 3.3 4.1 3.5 4.4 6.1 5.8 5.5 20:1iso 0.4 0.3 0.3 0.3 0.3 0.3 0.0 0.5 0.6 0.5 0.5 20:2ω6 0.8 0.8 0.9 0.8 0.6 0.8 0.7 1.3 1.5 1.4 1.4 20:3ω3 0.6 0.8 0.8 0.8 0.7 0.8 0.7 0.6 0.7 0.7 0.6 22:0 0.4 0.5 0.5 0.5 0.4 0.5 0.5 0.6 0.6 0.6 0.6 20:4ω3 0.2 0.3 0.3 0.3 0.4 0.4 0.5 0.0 0.0 0.0 0.0 22:1 1.1 1.1 1.2 1.1 0.5 0.9 0.8 1.6 2.2 1.9 2.0 20:5ω3 0.7 1.3 1.6 1.5 1.6 1.1 1.7 0.0 0.0 0.0 0.1 22:2ω6 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.3 0.2 0.2 22:4ω6 + 22:3ω3 0.3 0.2 0.3 0.3 0.0 0.3 0.0 0.4 0.6 0.5 0.5 24:0 0.3 0.3 0.3 0.3 0.0 0.3 0.0 0.4 0.4 0.4 0.4 24:1 0.3 0.4 0.4 0.3 0.0 0.3 0.0 0.5 0.6 0.5 0.5 22:5ω3 0.3 1.1 1.2 1.1 1.1 0.9 0.8 0.0 0.0 0.0 0.0 22:6ω3 4.7 9.0 8.5 8.3 8.3 7.1 4.9 0.0 0.0 0.0 0.0

TABLE-US-00012 TABLE 12 Compiled data from the total seed lipid profiles from transgenic seed shown in Table 11. Calculations do not include the `minor fatty acids` in Table 11. FD5.46 Parameter pooled # 2 # 4 # 8 # 7 # 9 # 1 # 3 # 5 # 6 # 10 total w3 (% of total FA) 36.1 46 45.4 45 48.2 44.2 40.1 28.9 29.9 30.2 28.9 total w6 (% of total FA) 25.8 20.4 20.7 20.3 19.5 21.1 25 33.7 32.6 31.8 33.8 w3/w6 ratio 1.40 2.25 2.19 2.22 2.47 2.09 1.60 0.86 0.92 0.95 0.86 w6/w3 ratio 0.71 0.44 0.46 0.45 0.40 0.48 0.62 1.17 1.09 1.05 1.17 total novel w3 (% of total FA) 8.1 18.5 18 16.9 18.6 15.2 12 0 0 0 0.1 total novel w6 (% of total FA) 1.1 2.2 1.2 1.1 1 1 1.2 1.5 2.3 2 2.2 novel w3/w6 ratio 7.36 8.41 15.00 15.36 18.60 15.20 10.00 0.05 novel w6/w3 ratio 0.14 0.12 0.07 0.07 0.05 0.07 0.10 22.00 OA to EPA efficiency 8.2% 15.6% 15.5% 15.1% 15.1% 12.8% 10.5% 0.0% 0.0% 0.0% 0.1% OA to DHA efficiency 6.7% 12.3% 11.6% 11.5% 11.4% 10.0% 7.0% 0.0% 0.0% 0.0% 0.0% LA to EPA efficiency 9.2% 17.2% 17.1% 16.7% 16.2% 13.9% 11.4% 0.0% 0.0% 0.0% 0.2% LA to DHA efficiency 7.6% 13.6% 12.9% 12.7% 12.3% 10.9% 7.5% 0.0% 0.0% 0.0% 0.0% ALA to EPA efficiency 15.8% 24.8% 24.9% 24.2% 22.8% 20.6% 18.5% 0.0% 0.0% 0.0% 0.3% ALA to DHA efficiency 13.0% 19.6% 18.7% 18.4% 17.2% 16.1% 12.2% 0.0% 0.0% 0.0% 0.0% total saturates 17.6 17.8 17.8 17.6 18 17.8 18.1 18.2 17.7 17.8 18.1 total monounsaturates 19.8 15.5 16 16.6 14.3 16.6 16.8 18.7 19.3 19.6 18.6 total polyunsaturates 62.5 66.6 66.4 65.6 67.7 65.6 65.1 63 63.1 62.5 63.2 total C20 9.6 9.3 9.8 9.9 8.1 8.9 8.5 8.6 11 10.3 10.1 total C22 5.4 10.3 10 9.7 9.4 8.3 5.7 0.6 0.9 0.7 0.7 C20/C22 ratio 1.78 0.90 0.98 1.02 0.86 1.07 1.49 14.33 12.22 14.71 14.43

[0669] DHA was present in six of the 10 individual seeds. The four other seeds did not have DHA and were presumed to be null segregants which did not have the T-DNA, based on hemizygosity of the T-DNA insertion in the parental plant. Extracted lipid from the single seed with the highest level of DHA had 9.0% DHA while the sum of the percentages for EPA, DPA and DHA was 11.4%. The sum of the percentages for the new ω3 fatty acids produced in this seed as a result of the transformation (SDA, ETrA, ETA, EPA, DPA, DHA) was 19.3% whilst the corresponding sum for the new ω6 fatty acids (GLA, EDA, DGLA, ARA and any ω6 elongation products) was 2.2% only GLA and EDA were detected as new ω6 fatty acids. The total ω6 FA (including LA) to ω3 FA (including ALA) ratio was found to be 0.44. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA) ratio in the seed with the highest DHA level was 0.12. The level of total saturated fatty acids was about 17.8% and the level of monounsaturated fatty acids was about 15.5%. The level of total ω6-fatty acids was about 20.4% and the level of ω3-fatty acids was about 46%. Overall conversion efficiencies were calculated to be: OA to EPA=15.6%, OA to DHA=12.3%, LA to EPA=17.2%, LA to DHA=13.6%, ALA to EPA=24.8%, ALA to DHA=19.6%.

[0670] Homozygous seed from this line was obtained in the T4 generation. Up to 10.3% DHA was produced in event FD5-46-18-110 with an average of 7.3% DHA observed across the entire T4 generation.

[0671] Homozygous seed was planted out across several glasshouses to generate a total of over 600 individual plants. Oil is being extracted from the seed using a variety of methods including soxhlet, acetone and hexane extractions.

[0672] Since the number of independently transformed lines of C. sativa obtained as described above was low, further experiments to transform C. sativa with pJP3416-GA7 are performed. The inventors predict that DHA levels of greater than 10% as a percentage of total fatty acids in seed oil will be achieved in further transformed lines, and plants which are homozygous for the T-DNA to 20% DHA. Twenty C. sativa GA7_modH events were generated and seed is being analysed for DHA content. Three GA7_modB events were generated and analysis of the T1 seed from event CMD17.1 revealed a pooled seed DHA content of 9.8%. The highest single seed DHA value was found to be 13.5%.

Example 4

Stable Expression of Transgenic DHA Pathways in Brassica napus Seeds

[0673] B. napus Transformation and Analysis of Fatty Acid Composition Using Single Vector

[0674] The binary vector pJP3416-GA7 was used to generate transformed Brassica napus plants and seeds from the plants. The vector pJP3416-GA7 as described above was introduced into Agrobacterium tumefaciens strain AGL1 via standard electroporation procedures. Cultures of the transgenic Agrobacterium cells were grown overnight at 28° C. in LB medium with agitation at 150 rpm. The bacterial cells were collected by centrifugation at 4000 rpm for 5 minutes, washed with Winans AB medium (Winans, 1988) and re-suspended in 10 mL of Winans AB medium (pH 5.2) and growth continued overnight in the presence of kanamycin (50 mg/L), rifampicin (25 mg/L) and 100 μM acetosyringone. Two hours before infection of the Brassica cells, spermidine (120 mg/L) was added and the final density of the bacteria adjusted to an OD 600 nm of 0.3-0.4 with fresh AB media. Freshly isolated cotyledonary petioles from 8-day old Brassica napus seedlings grown on 1/2 MS (Murashige and Skoog, 1962) or hypocotyl segments preconditioned by 3-4 days on MS media with 1 mg/L thidiazuron (TDZ) and 0.1 mg/L α-naphthaleneacetic acid (NAA) were infected with 10 mL Agrobacterium cultures for 5 minutes. The explants infected with Agrobacterium were then blotted on sterile filter paper to remove the excess Agrobacterium and transferred to co-cultivation media (MS media with 1 mg/L TDZ, 0.1 mg/L NAA and 100 μM acetosyringone) supplemented with or without different antioxidants (L-cysteine 50 mg/L and ascorbic 15 mg/L). All the plates were sealed with parafilm and incubated in the dark at 23-24° C. for 48 hrs.

[0675] The treated explants were then washed with sterile distilled water containing 500 mg/L cefotaxime and 50 mg/L timentin for 10 minutes, rinsed in sterile distilled water for 10 minutes, blotted dry on sterile filter paper, transferred to pre-selection media (MS containing 1 mg/L TDZ, 0.1 mg/L NAA, 20 mg/L adenine sulphate (ADS), 1.5 mg/L AgNO₃, 250 mg/L cefotaxime and 50 mg/L timentin) and cultured for five days at 24° C. with a 16 h/8 h photoperiod. They were then transferred to selection media (MS containing 1 mg/L TDZ, 0.1 mg/L NAA, 20 mg/L ADS, 1.5 mg/L AgNO₃, 250 mg/L cefotaxime and 50 mg/L timentin) with 1.5 mg/L glufosinate ammonium as the agent for selection of transformed cells, and cultured for 4 weeks at 24° C. with 16 h/8 h photoperiod with a biweekly subculture on to the same media. Explants with green callus were transferred to shoot initiation media (MS containing 1 mg/L kinetin, 20 mg/L ADS, 1.5 mg/L AgNO₃, 250 mg/L cefotaxime, 50 mg/L timentin and 1.5 mg/L glufosinate ammonium) and cultured for another 2-3 weeks. Shoots emerging from the resistant explants were transferred to shoot elongation media (MS media with 0.1 mg/L gibberelic acid, 20 mg/L ADS, 1.5 mg/L AgNO₃, 250 mg/L cefotaxime and 1.5 mg/L glufosinate ammonium) and cultured for another two weeks. Healthy shoots 2-3 cm long were selected and transferred to rooting media (1/2 MS containing 1 mg/L NAA, 20 mg/L ADS, 1.5 mg/L AgNO₃ and 250 mg/L cefotaxime) and cultured for 2-3 weeks. Well established shoots with roots were transferred to pots containing seedling raising mix and grown in a growth cabinet for two weeks and subsequently transferred to a glasshouse. Approximately 40 (T₀) plants transformed with the GA7 construct were obtained by this method.

[0676] Plants were grown to maturity after being allowed to self-fertilise. Seeds obtained from transformed plants were analysed for fatty acid composition in their seedoil as described in Example 1. Data for a transformed line with the highest DHA level are shown in Table 13. DHA levels on average were significantly lower in the seedoil of the B. napus seeds transformed with the T-DNA from pJP3416-GA7 than in A. thaliana seeds (Example 2) or Camelina seeds (Example 3) transformed with the same construct. The highest level of DHA in approximately 40 lines was found to be 1.52% with the majority of the transgenic lines having detectable DHA. It was noted that there was a substantial accumulation of ALA, about 35% of the total fatty acids, in these seeds which was not being converted efficiently to SDA or following products in the pathway.

[0677] Fatty acid profile analysis of single B. napus seeds from a T₁ event, CT125-2, was performed to better determine the amount of DHA produced in transgenic seeds. Seeds were found to contain between 0% (null seeds) and 8.5% DHA (Table 13).

[0678] Some of the seeds from the plant line CT116 as well as other transgenic lines showing DHA production were sown to produce progeny plants. RT-PCR was performed on total RNA isolated from developing embryos from these plants in order to determine why the GA7 construct performed poorly for DHA production relative to transgenic A. thaliana and C. sativa having the same construct, and poorly relative to the combination of the genes on pJP3115 and pJP3116 (below). RT-PCR was performed on total RNA using a one-step RT-PCR kit (Invitrogen) and gene-specific primers targeting each transgene. This confirmed that each of the genes in the GA7 construct was expressed well in the B. napus transformants except for the Δ6-desaturase which was poorly expressed in the majority of transformed seeds. The other genes from this construct functioned well in both B. napus and A. thaliana seeds, for example the Δ12- and Δ15-desaturases which functioned to produce increased levels of LA and ALA in the seeds whilst decreasing oleic acid levels. A representative RT-PCR gel is shown in FIG. 7 which clearly shows the low expression of the Δ6-desaturase relative to the other transgenes from pJP3416-GA7.

[0679] Transgenic plants and seed which are homozygous for the transgenes are generated by planting out progeny from the lines with the highest DHA.

TABLE-US-00013 TABLE 13 Fatty acid composition as a percentage of total fatty acids in seedoil from independent T₁ Brassica napus seed transformed with pJP3416-GA7, lines CT116-11 and CT-125-2 compared to wild-type (untransformed) control. 22:6ω3 is DHA. Data from single CT125-2 B. napus seeds is denoted by `SS`. CT116- CT125- CT125-2 CT125-2 CT125-2 Control 11 2 #2 SS #3 SS #10 SS 14:0 0.1 0.2 0.1 0.1 0.1 0.1 16:0 4.3 7.2 5.2 6.5 4.7 7.7 16:1 0.2 0.5 0.4 0.3 0.3 0.8 16:3 0.2 0.2 0.2 0.1 0.2 0.2 18:0 2.1 2.2 2.4 2.3 2.3 2.8 18:1d9 59.1 27.0 38.1 34.0 19.3 14.8 18:1d11 3.7 6.6 4.2 4.4 4.3 9.6 18:2 19.7 14.1 16.6 13.9 10.2 10.2 18:3ω3 8.3 35.2 27.7 34.1 49.5 37.9 20:0 0.6 0.5 0.6 0.4 0.3 0.7 18:4ω3 0.0 0.9 0.3 0.5 0.6 2.6 20:1d11 1.2 1.1 1.0 1.0 0.8 0.6 20:1is0 0.2 0.1 0.2 20:2ω6 0.1 0.1 0.1 0.1 0.1 0.1 20:3ω3 1.3 0.7 0.8 1.6 0.9 22:0 0.3 0.4 0.3 0.1 0.1 0.4 20:4ω3 0.1 0.3 0.4 0.6 0.5 22:1 20:5ω3 0.1 0.3 22:3ω3 0.1 24:0 0.2 0.4 0.3 0.1 0.1 0.3 24:1 0.1 0.3 0.1 0.1 0.2 0.1 22:5ω3 0.1 0.1 0.1 0.1 0.5 22:6ω3 1.52 1.2 1.3 2.7 8.5

B. napus Transformation and Analysis of Fatty Acid Composition Using Two Vectors

[0680] In another experiment in B. napus and as an alternative format for introducing the transgenes, the binary vectors pJP3115 and pJP3116 as described in WO 2010/057246 were used to separately generate transformed B. napus plants and transformed seeds were obtained from the plants. The T-DNA on pJP3115 comprised chimeric genes encoding the Crepis palestina Δ12-desaturase, Micromonas pusilla Δ6-desaturase, Pyramimonas cordata Δ6-elongase and Pavlova salina Δ5-desaturase and the T-DNA on pJP3116 contained chimeric genes encoding Perilla frutescens Δ15-desaturase, Pyramimonas cordata Δ5-elongase and Pavlova salina Δ4-desaturase. The two T-DNAs, when present together and expressed in developing seeds, formed a 7-gene pathway for producing DHA from endogenously produced oleic acid. These vectors were introduced into Agrobacterium tumefaciens strain AGL1 via standard electroporation procedures and the transformed cells used independently to transform B. napus using the method as described above to generate stably transformed T₀ plants. 29 pJP3115 and 19 pJP3116 transformants were obtained and these plants were grown to maturity and seeds obtained after self-fertilisation were analysed for fatty acid composition in their seedoil. Transformation with the T-DNA from pJP3115 was expected to result in EPA production from endogenously produced ALA whilst transformation with the T-DNA from pJP3116 was expected to result in increased ALA production from LA. Several plants were identified which displayed these phenotypes. The majority of events displayed a decreased OA/increased LA phenotype due to Δ12 desaturation with a low level of EPA production. Up to 2.6% EPA was observed in pJP31115 transgenic pooled seed. Similarly, the majority of pJP3116 events were found to have an elevated ALA phenotype due to Δ15-desaturase activity. Up to 18.5% ALA was found in pooled seed transformed with the T-DNA from pJP3116.

[0681] T₁ plants from the lines with the highest levels of EPA and ALA were crossed and the progeny seed (F1) from 24 recovered events analysed for DHA content. DHA was found in 17 of these events with up to 1.9% DHA found in pooled seed from these events. Single-seed analysis was performed to determine the range of DHA production--the data are shown in Table 14. A large range of DHA levels were observed in the crossed progeny, probably due to the hemizygous nature of the T-DNAs in the parental plants, so that some seeds did not receive both T-DNAs. Up to 6.7% DHA was observed in total seed lipid.

TABLE-US-00014 TABLE 14 Fatty acid composition as a percentage of total fatty acids in seedoil from B. napus F1 single seeds that were from a cross of plants transgenic for the T-DNA from pJP3115 with plants transgenic for the T-DNA from pJP3116. B1, B2 and B4 designate events. 0.0 = not detectable by the GC method. B1.1 B1.2 B1.3 B1.4-g B1.5-g B2.1 B2.2 B2.3g B2.4g B2.5g B3.1 B3.2 14:0 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.2 0.2 0.1 0.1 0.1 16:0 6.6 6.4 4.5 12.3 7.9 5.1 5.0 10.1 8.5 6.8 5.3 7.2 16:1 0.4 0.5 0.2 1.0 0.6 0.4 0.4 0.6 1.1 0.5 0.5 0.6 16:3 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.2 18:0 2.3 2.6 2.2 1.6 2.9 2.9 3.4 2.2 1.8 2.9 3.4 2.4 18:1 34.1 39.3 46.9 14.9 20.7 41.6 46.3 14.4 23.4 38.3 43.6 32.0 18:1d11 4.6 5.8 2.7 6.8 6.2 3.8 4.9 5.9 8.7 4.5 5.5 5.1 18:2 33.6 30.7 30.4 29.2 34.4 31.7 27.7 33.2 23.9 33.3 27.9 33.4 18:3ω6 0.2 0.3 0.1 0.4 0.4 0.2 0.2 0.7 0.1 0.2 0.2 0.3 18:3ω3 10.3 7.1 7.7 18.7 14.9 8.2 5.9 14.8 28.1 6.3 7.3 10.0 20:0 0.6 0.7 0.6 0.5 0.7 0.8 0.9 0.6 0.4 0.7 0.9 0.7 18:4ω3 0.2 0.1 0.1 0.8 0.5 0.2 0.2 0.8 0.0 0.2 0.2 0.2 20:1d11 1.0 1.1 1.1 0.7 0.8 1.1 1.1 0.5 0.9 1.1 1.1 0.9 20:1iso 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.3 0.1 0.1 0.1 20:2ω6 0.4 0.3 0.2 0.5 0.5 0.4 0.3 0.4 0.5 0.5 0.3 0.5 20:3ω6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20:4ω6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 20:3ω3 1.8 1.6 1.1 2.8 2.1 1.1 1.0 2.7 0.7 1.4 0.9 1.6 22:0 0.3 0.4 0.3 0.3 0.4 0.4 0.5 0.3 0.3 0.4 0.5 0.4 20:4ω3 0.3 0.2 0.2 0.4 0.4 0.1 0.1 0.5 0.0 0.2 0.1 0.2 22:1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20:5ω3 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.2 0.0 0.0 0.0 0.0 22:2ω6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 22:4ω6 0.1 0.2 0.1 0.2 0.2 0.1 0.1 0.4 0.2 0.2 0.1 0.2 24:0 0.3 0.4 0.2 0.2 0.3 0.3 0.3 0.3 0.4 0.4 0.4 0.3 22:5ω6 0.1 0.2 0.1 0.2 0.3 0.1 0.1 0.5 0.0 0.2 0.1 0.2 24:1 0.2 0.2 0.2 0.3 0.3 0.2 0.2 0.3 0.3 0.2 0.2 0.2 22:5ω3 0.7 0.7 0.3 2.1 1.6 0.3 0.4 3.2 0.0 0.5 0.4 1.2 22:6ω3 1.4 1.0 0.5 5.5 3.9 0.8 0.7 6.7 0.0 1.1 0.8 2.0

TABLE-US-00015 TABLE 15 Compiled data from the total seed lipid profiles from transgenic seed shown in Table 14. Calculations do not include the `minor fatty acids` in Table 14. Parameter B1.1 B1.2 B1.3 B1.4-g B1.5-g B2.1 B2.2 B2.3g B2.4g B2.5g B3.1 total w3 (% of total FA) 4.6 3.9 2.3 12.1 9 2.7 2.6 14.8 0.8 3.6 2.6 total w6 (% of total FA) 44.5 38.5 38.5 48.8 50.3 40.5 34.1 49.4 52.7 40.5 35.7 w3/w6 ratio 0.10 0.10 0.06 0.25 0.18 0.07 0.08 0.30 0.02 0.09 0.07 w6/w3 ratio 9.67 9.87 16.74 4.03 5.59 15.00 13.12 3.34 65.88 11.25 13.73 total novel w3 (% of total FA) 2.6 2 1.1 8.9 6.5 1.4 1.4 11.4 0 2 1.5 total novel w6 (% of total FA) 10.5 7.5 7.9 19.1 15.4 8.4 6.1 15.8 28.3 6.7 7.5 novel w3/w6 ratio 0.25 0.27 0.14 0.47 0.42 0.17 0.23 0.72 0.00 0.30 0.20 novel w6/w3 ratio 4.04 3.75 7.18 2.15 2.37 6.00 4.36 1.39 3.35 5.00 OA to EPA efficiency 2.5% 2.1% 0.9% 10.1% 6.9% 1.3% 1.3% 12.8% 1.9% 1.4% OA to DHA efficiency 1.7% 1.2% 0.6% 7.2% 4.8% 0.9% 0.8% 8.5% 1.3% 1.0% LA to EPA efficiency 4.3% 4.0% 2.0% 12.6% 9.4% 2.5% 3.0% 15.7% 3.6% 3.1% LA to DHA efficiency 2.9% 2.4% 1.2% 9.0% 6.6% 1.9% 1.9% 10.4% 2.5% 2.1% ALA to EPA efficiency 47.7% 44.7% 36.4% 68.1% 65.9% 44.0% 45.8% 72.1% 47.1% 50.0% ALA to DHA efficiency 31.8% 26.3% 22.7% 48.7% 45.9% 32.0% 29.2% 47.9% 32.4% 33.3% total saturates 10.2 10.6 7.9 15.1 12.4 9.6 10.2 13.7 11.6 11.3 10.6 total monounsaturates 40.4 47 51.1 23.8 28.7 47.2 53 21.8 34.7 44.7 51 total polyunsaturates 49.2 42.5 40.9 61 59.4 43.3 36.8 64.3 53.7 44.2 38.4 total C20 4.2 4 3.2 5.1 4.7 3.6 3.5 5.1 2.8 4 3.4 total C22 2.6 2.5 1.3 8.3 6.4 1.7 1.8 11.1 0.5 2.4 1.9 C20/C22 ratio 1.62 1.60 2.46 0.61 0.73 2.12 1.94 0.46 5.60 1.67 1.79

[0682] Compiled data from the total lipid profiles (Table 14) are shown in Table 15. From the data in Table 15, the total ω6 FA (including LA) to ω3 FA (including ALA) ratio in the seed with the highest level of DHA was 3.34. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA) ratio was 1.39. The level of total saturated fatty acids was about 13.7% and the level of monounsaturated fatty acids was about 21.8%. The level of total ω6-fatty acids was about 46.4% and the level of ω3-fatty acids was about 14.8%. Overall conversion efficiencies were calculated to be: OA to EPA=12.8%, OA to DHA=8.5%, LA to EPA=15.7%, LA to DHA=10.4%, ALA to EPA=72.1%, ALA to DHA=47.9%. The reduced efficiency of the ω6 fatty acids to ω3 fatty acids conversion observed in this experiment with the combination of the pJP3115 and pJP3116 was thought to be due to a lower efficiency of the plant Δ15-desaturase compared to the fungal Δ15/ω3 desaturase (Examples 2 and 3) when combined with the genes for conversion of ALA to DHA.

[0683] Progeny from DHA-containing lines which are homozygous for all of the introduced transgenes are generated for analysis.

Example 5

Modifications to T-DNAs Encoding DHA Pathways in Plant Seeds

[0684] In order to improve the DHA production level in B. napus beyond the levels described in Example 4, the binary vectors pJP3416-GA7-modA, pJP3416-GA7-modB, pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7-modE and pJP3416-GA7-modF were constructed as follows. These binary vectors were variants of the pJP3416-GA7 construct described in Example 2 and were designed to further increase the synthesis of DHA in plant seeds, particularly by improving Δ6-desaturase and Δ6-elongase functions. SDA had been observed to accumulate in some seed transformed with the GA7 construct due to a relatively low elongation efficiency compared to the Δ5-elongase, so amongst other modifications, the two elongase gene positions were switched in the T-DNA.

[0685] The two elongase coding sequences in pJP3416-GA7 were switched in their positions on the T-DNA to yield pJP3416-GA7-modA by first cloning a new P. cordata Δ6-elongase cassette between the SbfI sites of pJP3416-GA7 to replace the P. cordata Δ5-elongase cassette. This construct was further modified by exchanging the FP1 promoter driving the M. pusilla Δ6-desaturase with a conlinin Cnl2 promoter (pLuCnl2) to yield pJP3416-GA7-modB. This modification was made in an attempt to increase the Δ6-desaturase expression and thereby enzyme efficiency. It was thought that the Cnl2 promoter might yield higher expression of the transgene in B. napus than the truncated napin promoter. pJP3416-GA7-modC was produced by adding a second M. pusilla Δ6-desaturase cassette with slightly different codon usage (SEQ ID NO: 15) and driven by the FP1 promoter, which was inserted at the PmeI site just inside the right border of pJP3416-GA7-modB. The second Δ6-desaturase cassette was added to both pJP3416-GA7-modB and pJP3416-GA7-modF in order to increase the Δ6-desaturase expression level and extend the time period during seed development for expression of Δ6-desaturase by the use of multiple promoters. Different codon usages were used in the two nucleotide sequences to result in the translation of the same protein sequence without risking co-suppression from similar coding regions within the same T-DNA. pJP3416-GA7-modD and pJP3416-GA7-modE were similar variants in which a third MAR sequence, corresponding to nucleotides 16649-17816 of SEQ ID NO: 1, was added to pJP3416-GA7 and pJP3416-GA7-modB, respectively, at the PmeI site. pJP3416-GA7-modF was produced by adding a second M. pusilla Δ6-desaturase cassette containing the native Δ6-desaturase nucleotide sequence and driven by the FP1 promoter at the PmeI site at the right border of pJP3416-GA7-modB. pJP3416-GA7-modG was made by first replacing the M. pusilla Δ6-desaturase cassette with a Cnl2:P. cordata Δ5-elongase cassette by restriction cloning at the AscI-PacI sites. pJP3416-GA7-modG was then made by replacing the original FAE1:P. cordata Δ5-elongase cassette with a FAE1:M. pusilla Δ6-desaturase cassette by restriction cloning at the SbfI sites. The nucleotide sequences of the T-DNAs from each of these genetic constructs are shown as: pJP3416-GA7-modB (SEQ ID NO:2), pJP3416-GA7-modC (SEQ ID NO:3), pJP3416-GA7-modD (SEQ ID NO:4), pJP3416-GA7-modE (SEQ ID NO:5), pJP3416-GA7-modF (SEQ ID NO:6) and pJP3416-GA7-modG (SEQ ID NO:7).

[0686] The binary vectors pJP3416-GA7-modB, pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7-modE, pJP3416-GA7-modF and pJP3416-GA7-modG are used to generate transformed Brassica somatic embryos and Brassica napus, Camelina sativa and Arabidopsis thaliana plants and progeny seeds. Data for pJP3416-GA7-modB are shown in the next Example.

[0687] Eight transgenic pJP3416-GA7-modB A. thaliana events and 15 transgenic pJP3416-GA7-modG A. thaliana events were generated. Between 3.4% and 7.2% DHA in pooled pJP3416-GA7-modB seed was observed and between 0.6 and 4.1% DHA in pooled T2 pJP3416-GA7-modG seed was observed. Several of the highest pJP3416-GA7-modB events were sown out on selectable media and surviving seedlings taken to the next generation. Seed is being analysed for DHA content. Since the pooled T1 seeds represented populations that were segregating for the transgenes and included any null segregants, it is expected that the homozygous seeds from progeny plants will have increased levels of DHA, up to 20% of the total fatty acid content in the seed oil. The other modified constructs were used to transform A. thaliana. Although only a small number of transformed lines were obtained, none yielded higher levels of DHA than the modB construct.

[0688] The pJP3416-GA7-modB construct was also used to generate transformed B. napus plants of cultivar Oscar and in a breeding line designated NX005. Ten independent transformed plants (T0) were obtained so far for the Oscar transformation, and 20 independent lines for NX005. Seed (T1 seed) was harvested from these transgenic lines. Pools of seed were tested for levels of DHA in the seed oil, and two lines which showed the highest levels were selected, these were designated lines CT132.5 (in cultivar Oscar) and CT133.15 (in NX005). Twenty seeds from CT132.5 and 11 seeds from CT133.15 were imbibed and, after two days, oil was extracted from a half cotyledon from each of the individual seeds. The other half cotyledons with embryonic axes were kept and cultured on media to maintain the specific progeny lines. The fatty acid composition in the oil was determined; the data is shown in Table 16 for CT132.5. The DHA level in ten of the 20 seeds analysed was in the range of 7-20% of the total fatty acid content as determined by the GC analysis. Other seeds had less than 7% DHA and may have contained a partial (incomplete) copy of the T-DNA from pJP3416-GA7-modB. The transgenic line appeared to contain multiple transgene insertions that were genetically unlinked. The seeds of transgenic line CT133.15 exhibited DHA levels in the range 0-5%. Seeds with no DHA were likely to be null segregants. These data confirmed that the modB construct performed well for DHA production in canola seed.

[0689] The pJP3416-GA7-modB and pJP3416-GA7-modF constructs were also used to generate transformed Camelina sativa plants. At least 24 independent transformed plants (T0) were obtained and examined in more detail by progeny analysis. Seed (T1 seed) was harvested from these transgenic lines. Pools of seed were tested for levels of DHA in the seed oil, and 6 lines which showed the highest levels of DHA (between 6% and 9%) were selected. The DHA levels in 20 T1 seeds from each line were analysed-most seeds exhibited DHA levels in the range of 6-14% of the total fatty acid content as determined by the GC analysis. The fatty acid composition in the oil was determined; the data is shown in Table 17 for several transgenic seeds. These data confirmed that the modB and modF constructs both performed well for DHA production in Camelina seed.

TABLE-US-00016 TABLE 16 Fatty acid profiles of half cotyledons of germinating T1 transgenic B. napus seeds containing the modB construct. Up to 18.1% DHA was observed with numerous samples containing greater than 10% DHA. Seed 14:0 16:0 16:1d3? 16:1 16:3 18:0 18:1 18:1d11 18:2 18:3n6 18:3n3 20:0 18:4n3 C20:1d11 1 0.1 4.2 0.1 0.1 0.2 1.8 29.9 2.5 9.9 0.1 38.4 0.5 0.8 1.0 2 0.1 4.7 0.1 0.1 0.2 4.0 23.0 2.3 7.4 0.3 29.3 1.0 4.3 1.1 3 0.1 3.7 0.2 0.1 0.2 1.8 55.1 1.9 4.7 0.2 15.2 0.8 1.8 1.4 4 0.1 4.6 0.2 0.2 0.2 2.9 22.1 1.8 6.6 0.4 26.5 1.0 7.2 1.0 5 0.1 4.0 0.1 0.1 0.2 1.7 27.4 2.1 8.1 0.3 26.4 0.6 2.8 1.0 6 0.1 3.5 0.1 0.1 0.2 1.6 59.8 2.0 4.3 0.1 18.5 0.6 0.5 1.3 7 0.1 6.0 0.3 0.3 0.3 1.7 16.6 2.6 23.9 1.0 23.2 0.6 5.4 0.8 8 0.1 4.9 0.1 0.1 0.2 2.7 12.9 1.4 11.7 0.3 34.3 0.9 5.0 0.9 9 0.1 3.9 0.1 0.1 0.1 2.4 41.6 1.7 21.5 0.0 23.4 0.7 0.0 1.2 10 0.1 3.7 0.2 0.1 0.1 2.1 30.9 1.7 19.2 0.4 23.6 0.7 2.1 1.1 11 0.1 5.7 0.4 0.3 0.2 3.8 41.2 2.4 26.7 2.1 7.2 1.3 0.3 1.2 12 0.1 4.6 0.0 0.1 0.2 2.4 25.5 1.7 16.1 0.3 28.9 0.8 3.9 1.1 13 0.1 4.3 0.1 0.1 0.1 4.2 19.4 1.6 9.2 0.1 45.5 1.0 0.2 1.1 14 0.1 6.3 0.2 0.2 0.2 4.0 10.5 2.3 8.4 0.3 31.1 1.3 3.9 0.8 15 0.1 5.1 0.1 0.2 0.2 3.3 16.8 2.4 11.2 0.3 28.8 1.0 4.5 0.9 16 0.1 4.4 0.1 0.1 0.2 4.0 16.2 1.5 11.6 0.2 33.5 0.9 2.8 1.1 17 0.2 7.2 0.2 0.2 0.2 4.9 15.0 2.1 8.9 0.3 25.9 1.4 5.1 0.9 18 0.1 4.0 0.1 0.1 0.2 2.3 64.8 1.2 7.2 0.1 12.5 1.0 3.5 1.5 19 0.1 3.9 0.1 0.1 0.2 4.6 36.9 1.7 7.1 0.2 28.6 1.2 1.8 1.2 20 0.1 4.8 0.1 0.1 0.2 6.0 18.5 1.2 12.8 0.2 34.8 1.4 2.4 1.1 Seed 20:1d13 C20:2n6 C20:3n3 C22:0 20:4n3 20:5n3 22:3n3 C24:0 C24:1 22:5n3 C22:6n3 1 0.0 0.1 2.1 0.3 2.8 0.3 0.1 0.2 0.2 0.5 3.9 2 0.0 0.1 1.9 0.4 6.9 1.0 0.0 0.3 0.1 1.7 9.5 3 0.0 0.1 0.3 0.5 11.3 0.0 0.0 0.3 0.2 0.0 0.0 4 0.0 0.1 0.8 0.5 11.2 1.9 0.0 0.2 0.2 1.7 8.7 5 0.0 0.1 1.5 0.3 7.6 1.5 0.0 0.1 0.1 1.8 12.2 6 0.0 0.0 0.7 0.3 6.0 0.0 0.0 0.2 0.1 0.0 0.0 7 0.0 0.2 0.6 0.4 2.6 1.1 0.0 0.3 0.3 1.7 9.9 8 0.0 0.2 2.4 0.5 4.1 1.3 0.0 0.2 0.2 1.8 13.8 9 0.0 0.1 2.2 0.4 0.0 0.0 0.1 0.3 0.2 0.0 0.0 10 0.0 0.1 1.5 0.4 3.6 0.6 0.0 0.2 0.1 0.7 6.9 11 0.0 0.2 0.3 0.8 4.8 0.0 0.0 0.6 0.3 0.0 0.0 12 0.0 0.1 1.9 0.4 3.9 0.6 0.0 0.2 0.0 1.1 6.2 13 0.0 0.1 5.2 0.4 2.6 0.3 0.2 0.2 0.1 0.4 3.4 14 0.0 0.1 2.3 0.6 4.6 1.8 0.1 0.3 0.2 2.5 18.1 15 0.0 0.1 2.1 0.6 3.2 1.5 0.1 0.3 0.1 1.8 15.1 16 0.0 0.2 3.7 0.4 4.6 0.7 0.1 0.3 0.1 1.3 12.1 17 0.0 0.0 1.6 0.8 4.9 2.1 0.0 0.6 0.3 2.2 15.0 18 0.0 0.1 0.0 0.7 0.0 0.0 0.0 0.5 0.2 0.0 0.0 19 0.0 0.1 1.4 0.5 4.3 0.4 0.0 0.4 0.1 0.8 4.3 20 0.0 0.1 3.4 0.6 3.2 0.4 0.1 0.3 0.1 0.7 7.6

TABLE-US-00017 TABLE 17 Fatty acid profiles of T1 transgenic C. sativa seeds containing the modB or modF constructs C14:0 C16:0 C16:1 C18:0 C18:1 C18:1d11 C18:2 C18:3n6 C18:3n3 C20:0 18:4n3 123-8 0.1 7.3 0.0 5.2 7.9 1.0 7.7 0.7 29.9 2.3 6.0 123-12 0.1 8.3 0.0 5.3 7.2 1.2 8.7 0.9 27.2 2.5 5.7 5-8 0.1 8.3 0.1 3.5 9.4 1.3 8.1 1.1 29.0 1.0 9.3 5-9 0.1 8.1 0.0 3.5 9.4 1.2 8.4 1.2 29.2 1.0 9.0 17-10 0.1 8.7 0.1 4.1 8.4 1.3 5.5 1.2 26.1 1.6 11.8 17-26 0.1 8.8 0.1 5.5 5.0 1.3 7.6 0.9 27.8 2.7 10.1 C20:1d11 20:1d13 C20:2n6 C20:3n6 C20:4n6 C20:3n3 C22:0 20:4n3 C22:1 20:5n3 123-8 7.1 0.4 0.7 0.0 0.0 0.9 0.4 1.3 1.0 4.6 123-12 6.9 0.5 0.7 0.0 0.1 0.9 0.5 1.5 1.2 5.0 5-8 7.9 0.4 0.6 0.0 0.0 0.8 0.2 0.4 0.8 3.4 5-9 8.1 0.3 0.6 0.0 0.0 0.8 0.2 0.5 0.8 3.5 17-10 7.2 0.3 0.0 0.4 0.03 0.8 0.3 0.4 0.7 5.5 17-26 6.2 0.3 0.0 0.7 0.03 1.1 0.6 0.5 1.0 4.7 C22:2n6 22:3n3 C24:0 C24:1 22:5n3 C22:6n3 123-8 0.0 0.1 0.2 0.3 1.5 13.3 123-12 0.0 0.1 0.2 0.4 1.5 13.2 5-8 0.0 0.1 0.2 0.4 0.9 12.6 5-9 0.0 0.1 0.1 0.3 0.9 12.6 17-10 0.0 0.0 0.2 0.3 1.3 13.5 17-26 0.1 0.1 0.3 0.4 1.0 13.1

[0690] The inventors considered that, in general, the efficiency of rate-limiting enzyme activities in the DHA pathway can be greater in multicopy T-DNA transformants compared to single-copy T-DNA transformants, or can be increased by inserting into the T-DNA multiple genes encoding the enzyme which might be limiting in the pathway. Evidence for the possible importance of multi-copy transformants was seen in the Arabidopsis seeds transformed with the GA7 construct (Example 2), where the highest yielding DHA event had three T-DNAs inserted into the host genome. The multiple genes can be identical, or preferably are different variants that encode the same polypeptide, or are under the control of different promoters which have overlapping expression patterns. For example, increased expression could be achieved by expression of multiple Δ6-desaturase coding regions, even where the same protein is produced. In pJP3416-GA7-modF and pJP3416-GA7-modC, for instance, two versions of the M. pusilla Δ6-desaturase were present and expressed by different promoters. The coding sequences had different codon usage and therefore different nucleotide sequences, to reduce potential silencing or co-suppression effects but resulting in the production of the same protein.

Example 6

Activity of Seed-Specific Constructs in Somatic Embryos

[0691] In order to establish a rapid assay system which was predictive of expression of genetic constructs in seeds under the control of seed-specific promoters, a somatic embryo system was set up for Brassica napus. This used a vector to express the LEC2 transcription factor which is involved in initiation of somatic embryogenesis. As a demonstration, the binary vectors 35S:LEC2 and pJP107 (Petrie et al., 2010a and b) were introduced into Agrobacterium tumefaciens strain AGL1 via standard electroporation and the Agrobacterium transformants used to co-transform Brassica napus by co-cultivation. The T-DNA region of pJP107 contained genes encoding the Isochrysis galbana Δ9-elongase, P. salina Δ8-desaturase and P. salina Δ5-desaturase with each gene expressed by a seed-specific promoter. A control transformation used the 35S:LEC2 vector alone. 35S:LEC2 expression resulted in the generation of somatic embryos in tissue culture directly from the transformed B. napus callus tissue as described in Example 1.

[0692] Fatty acid analysis showed that the seed-specific genes on the T-DNA of the construct pJP107 were expressed in the transgenic somatic embryos in the presence of the co-transformed LEC2 gene and functioned to produce ARA (20:4.sup.Δ5, 8, 11, 14) from LA and EPA (20:5.sup.Δ5, 8, 11, 14, 17) from ALA. The data for three co-transformed somatic embryos are shown in Table 18 and the fatty acid composition of each compared to the fatty acid composition of seed oil from Brassica napus seed which was transgenic for, and expressing, the T-DNA of pJP107 (Petrie et al, 2010a and b). Similar total percentages of ARA and the intermediate fatty acids EDA (20:2ω6) and DGLA (20:3ω6), as well as conversion efficiencies, were observed in somatic embryo tissue when compared with stably-transformed seed profiles. Similar results were observed in the fatty acid compositions of the stable T₂ transgenic seed and somatic embryos: ω6 fatty acids were at a level of 26.6% and 25.6% (on average), respectively, whilst ARA levels were found to be 9.7% and 10.6% (on average), respectively.

[0693] When 35S:LEC2 alone was introduced and the somatic embryos analysed in a time-course, the fatty acid profile was found to change to a more embryo-like profile with 18:3.sup.Δ9, 12, 15 decreasing and 18:1.sup.Δ9 increasing in an inversely correlated manner (FIG. 8). These results indicated that the somatic embryos were indeed becoming seed-like in character and the genes on the T-DNA from pJP107 were expressed. This demonstrated that the somatic embryo system allowed a rapid characterisation of transgenic seed-specific constructs in B. napus without requiring the full process of producing a transgenic plant and, from that, mature seed.

TABLE-US-00018 TABLE 18 Fatty acid composition of lipid obtained from Brassica napus somatic embryos generated by co-transforming pJP107 with 35S:LEC2, compared to the control untransformed (WT) and T₂ seeds transformed with pJP107. Individual enzymatic conversion efficiencies are shown in brackets after the relevant enzymatic steps. D9-Elo is Δ9-elongase, D8-Des is Δ8-desaturase and D5-Des is Δ5-desaturase. WT T2 pJP107 transgenic seed LEC2:#45 LEC2:#57 LEC2:#58 18:1.sup.Δ9 57.2 45.7 3.8 2.5 1.9 18:2.sup.Δ9,12 19.1 8.7 10 10.6 10 18:3.sup.Δ9,12, 15 10.2 4.1 22.5 27.5 24.2 20:2.sup.Δ11,14 7.1 ± 1.9 (67% D9-elo) 5.2 (61.8% D9-elo) 3.7 (56.7% D9-elo) 4.6 (61.8% D9-elo) 20:3.sup.Δ8,11,14 1.1 ± 0.2 (60% D8-des) 0.4 (67% D8-des) 0.2 (73% D8-des) 0.4 (73% D8-des) 20:4.sup.Δ5,8,11,14 9.7 ± 0.9 (90% D5-des) 10.6 (98% D5-des) 10 (96% D5-des) 11.2 (97% D5-des) 20:3.sup.Δ11,14,17 4.0 ± 0.8 9.9 5.5 7.3 20:4.sup.Δ8,11,14,17 0.3 ± 0.1 0.4 0.3 0.4 20:5.sup.Δ5,8,11,14,17 2.4 ± 0.2 7.6 6.4 7.9 Total new 24.6 34.1 26.1 31.8

[0694] Using the same system to generate somatic embryos, Brassica napus cells were transformed separately with pJP3416-GA7-modB and pJP3416-GA7-modD. 42 embryos were obtained, 18 for modB and 24 for modD. Total lipid was extracted from the embryos and analysed for fatty acid composition. The embryos contained between 0% and up to 16.9% DHA (Table 19). The results with 0% DHA was presumed to be due to integration of only a partial T-DNA or an insertion into a transcriptionally silent region of the genome. The total ω3 FA (including ALA) to total ω6 FA (including LA) ratio was found to be 2.3 for embryo #270 and 11.96 for embryo #284. The total ω6 FA (including LA) to total (3 FA (including ALA) ratio was 0.08 for #284. The new ω6 FA (excluding LA) to new ω3 FA (excluding ALA) ratio was 0.03 for #284. Overall conversion efficiencies were calculated to be: (for embryos #270, #284) OA to EPA=14.0%, 29.8%; OA to DHA=9.7%, 24.2%; LA to EPA=15.4%, 30.7%; LA to DHA=10.7%, 25.0%; ALA to EPA=22.1%, 33.3%; ALA to DHA=15.3%, 27.0%. These efficiencies were similar, or greater than in the case of #284, to those observed for the T₃ pJP3416-GA7 Arabidopsis lines which indicated that the pJP3416-GA7-modB vector was capable of functioning well in B. napus cells. The SDA level was below 3.0%, indicating that the Δ6-elongase was performing even better than the GA7 construct. The individual enzyme efficiencies achieved in #284 were: Δ12-desaturase, 97.4%; ω3-desaturase, 92.3%; Δ6-desaturase, 38.2%; Δ6-elongase, 88.2%; Δ5-desaturase, 98.8%; Δ5-elongase, 94.1%; and Δ4-desaturase, 86.3%. Total saturates were 21.2%, total monounsaturates were 10.2%, total polyunsaturates were 68.6%.

[0695] The inventors believe this was the highest level of DHA achieved in B. napus cells to date, except for further data described below. This also demonstrated that the modification in pJP3416-GA7-modB relative to pJP3416-GA7 was effective in increasing the level of expression of the Δ6-desaturase gene. The binary vectors pJP3416-GA7, pJP3416-GA7-modA, pJP3416-GA7-modC, pJP3416-GA7-modD, pJP3416-GA7-modE and pJP3416-GA7-modF as described above are co-transformed with 35S:LEC2 to generate transformed B. napus somatic embryos. Up to 7.0% DHA was observed in modD embryos, 9.9% in modE embryos, 8.3% in modF embryos and 3.6% in a small number of modG embryos.

TABLE-US-00019 TABLE 19 Fatty acid composition of oil from Brassica napes somatic embryos #270 and #284 generated by co-transforming the seed-specific DHA acid construct pJP3416-GA7-modB with 35S:LEC2, and #286 and #289 (pJP3416-GA7-modD). #270 #284 #286 #289 14:0 0.3 0.2 0.2 0.2 16:0 14.0 15.7 17.2 16.6 16:1d9 0.7 0.4 0.8 0.8 16:3 0.5 0.6 1.1 1.3 18:0 2.6 2.4 2.5 2.5 18:1d9 6.6 1.8 1.5 1.1 18:1d11 6.3 6.8 6.5 6.7 18:2 18.9 4.5 10.0 9.8 18:3ω6 0.7 0.8 0.3 0.3 18:3ω3 33.0 37.2 42.0 41.5 20:0 0.9 0.9 0.8 0.8 18:4ω3 1.9 2.8 3.6 4.5 20:1d11 0.2 0.1 0.1 0.1 20:2ω6 0.1 0.1 0.1 0.2 20:3ω3 0.5 0.0 0.5 0.6 22:0 0.8 1.5 0.6 0.7 20:4ω3 0.2 0.9 0.7 0.7 20:5ω3 0.7 0.2 0.3 0.3 22:2ω6 0.0 1.2 0.0 0.0 22:3ω3 0.0 0.1 0.0 0.1 24:0 0.8 1.0 1.0 1.0 24:1 0.8 1.0 0.7 0.9 22:5ω3 2.4 2.7 3.2 3.0 22:6ω3 7.0 16.9 6.1 6.4

Example 7

Analysis of TAG from Transgenic A. thaliana Seeds Producing DHA

[0696] The positional distribution of DHA on the TAG from the transformed A. thaliana seed was determined by NMR. Total lipid was extracted from approximately 200 mg of seed by first crushing them under hexane before transferring the crushed seed to a glass tube containing 10 mL hexane. The tube was warmed at approximately 55° C. in a water bath and then vortexed and centrifuged. The hexane solution was removed and the procedure repeated with a further 4×10 mL. The extracts were combined, concentrated by rotary evaporation and the TAG in the extracted lipid purified away from polar lipids by passage through a short silica column using 20 mL of 7% diethyl ether in hexane. Acyl group positional distributions on the purified TAG were determined quantitatively as previously described (Petrie et al., 2010a and b).

[0697] The analysis showed that the majority of the DHA in the total seed oil was located at the sn-1/3 positions of TAG with little found at the sn-2 position (FIG. 9). This was in contrast to TAG from ARA producing seeds which demonstrated that 50% of the ARA (20:4.sup.Δ5, 8, 11, 14) was located at the sn-2 position of transgenic canola oil whereas only 33% would be expected in a random distribution (Petrie et al., 2012).

[0698] Positional distribution of DHA in the TAG from the B. napus seeds transformed with pJP3416-GA7 or with the combination of pJP3115 and pJP3116 is determined by essentially the same method.

[0699] The total lipid from transgenic A. thaliana seeds was also analysed by triple quadrupole LC-MS to determine the major DHA-containing triacylglycerol (TAG) species (FIG. 10). The most abundant DHA-containing TAG species was found to be DHA-18:3-18:3 (TAG 58:12; nomenclature not descriptive of positional distribution) with the second-most abundant being DHA-18:3-18:2 (TAG 58:11). Tri-DHA TAG (TAG 66:18) was observed in total seed oil, albeit at low but detectable levels. Other major DHA-containing TAG species included DHA-34:3 (TAG 56:9), DHA-36:3 (TAG 58:9), DHA-36:4 (TAG 58:10), DHA-36:7 (TAG 58:13) and DHA-38:4 (TAG 60:10). The identities of the two major DHA-containing TAG were further confirmed by Q-TOF MS/MS.

Example 8

Predicting DHA Production in B. napus Seeds

[0700] Efficient production of DHA in Arabidopsis seeds at a 15% level using the GA7 genetic construct was demonstrated in Example 2. The same construct in Brassica napus seeds produced only about 1.5% DHA in many (but not all) of the transformants, primarily due to the poor expression of the Δ6-desaturase gene of GA7 in this species (Example 4). Based on the realisation that modifications to the GA7 construct would overcome the Δ6-desaturase gene expression problem (see Example 5, as demonstrated in Example 6), calculations were performed to determine the likely fatty acid profile of B. napus transgenic seeds expressing the genes from a variant of pJP3416-GA7, where each transgene-encoded enzyme was performing as efficiently as was observed in A. thaliana with the GA7 construct. The predicted fatty acid compositions for three calculations (#1, #2, #3) are shown in Table 20. This was based on a wild-type (non-transformed) fatty acid composition for B. napus that included 59% oleic acid, 20% LA and 8% ALA. The three predicted partial fatty acid profiles shown in the lower half of the table were based on the conversion efficiencies for each enzymatic step shown in the upper half of the table. In prediction #2, a combination of Δ12-desaturation at 75% efficiency, Δ15-desaturation at 75%, Δ6-desaturation at 35%, Δ6-elongation at 80%, Δ5-desaturation at 90%, Δ5-elongation at 90% and Δ4-desaturation at 90% would result in the production of approximately 10% DHA in a typical canola transgenic seed. These efficiencies were all lower or about equal to the individual efficiencies seen in Arabidopsis, so prediction #2 represented a conservative estimate. The conversion efficiencies listed in #3 were approximations based on the efficient conversions seen in A. thaliana transformed with pJP3416-GA7. DHA was predicted to be produced at about 15% of the total fatty acid content in seedoil produced in B. napus seed, a result that mirrored the most efficient production levels observed in A. thaliana. Insertion of multiple T-DNAs in the homozygous state is expected to raise the DHA level to 20% in B. napus.

TABLE-US-00020 TABLE 20 Predicted fatty acid composition for selected fatty acids as a percentage of total fatty acid content in seedoil from Brassica napus transformed with a DHA pathway construct, based on observed enzymatic efficiencies in transgenic Arabidopsis. The enzymes are listed in order in the pathway for producing DHA from oleic acid. des = desaturase, elo = elongase. Predicted fatty acid compositions #1, #2 and #3 are based on the efficiencies in the upper half of the Table. WT #1 #2 #3 Enzyme d12-des 70% 75% 80% d15-des 70% 75% 80% d6-des 30% 35% 40% (ω3) d6-elo 80% 80% 90% d5-des 80% 90% 90% d5-elo 80% 90% 90% d4-des 80% 90% 90% Fatty acid 18:1d9 59% 26% 22% 18% 18:2ω6 20% 19% 17% 14% 18:3ω6 1% 2% 3% 18:3ω3 8% 30% 32% 34% 18:4ω3 3% 3% 2% 20:4ω3 2% 1% 2% 20:5ω3 2% 1% 2% 22:5ω3 1% 1% 2% 22:6ω3 5% 10% 15%

Example 9

Stable Expression of a Transgenic EPA Pathway in Plant Leaf

Binary Vector Construction

[0701] A binary vector, pOREO4+11ABGBEC_Cowpea_EPA_insert (SEQ ID NO:8), was designed for introduction of a T-DNA into plants for the synthesis of EPA in leaf tissues. It contained chimeric genes encoding the enzymes: M. pusilla Δ6-desaturase (SEQ ID NO:16), P. cordata Δ6-elongase (SEQ ID NO:25) and P. salina Δ5-desaturase (SEQ ID NO:30), each under the control of the CaMV 35S and A. thaliana rubisco small subunit (SSU) promoters (FIG. 9). The binary vector was constructed by synthesising the region 199-10878 of SEQ ID 2 and cloning this into the recipient binary vector pORE04 (Coutu et al., 1997) at the BsiWI and KasI sites. The three fatty acid biosynthesis genes coded for the enzymes required to convert ALA, 18:3.sup.Δ9, 12, 15 to EPA, 20:5.sup.Δ5, 8, 11, 14, 17.

Transient Expression of EPA Construct in N. benthamiana Leaf Cells

[0702] To test that the construct was correct and would express the genes efficiently in leaf tissues, the chimeric vector pORE04+11ABGBEC_Cowpea_EPA_insert was introduced into A. tumefaciens strain AGL1. The chimeric vector 35S:p19 was also introduced into A. tumefaciens strain AGL1 as described in Example 1. Cells from cultures of these infiltrated into leaf tissue of Nicotiana benthamiana plants in a 24° C. growth room. Several direct comparisons were infiltrated with the samples being compared located on either side of the same leaf. Experiments were performed in triplicate. Following infiltration, the plants were grown for a further five days before leaf discs were taken for fatty acid profile analysis by GC as described in Example 1. GC analysis revealed that the EPA vector was functioning to produce EPA in Nicotiana benthamiana leaf (Table 21) with the highest level of EPA found to be 10.7% of total leaf lipids.

Nicotiana tabacum Stable Transformation

[0703] The chimeric vector pORE04+11ABGBEC_Cowpea_EPA_insert was used to stably transform Nicotiana tabacum. The vector was introduced into A. tumefaciens strain AGL1 via standard electroporation procedure. The transformed cells were grown on solid LB media supplemented with kanamycin (50 mg/L) and rifampicin (25 mg/L) and incubated at 28° C. for two days. A single colony was used to initiate fresh culture. Following 48 h vigorous culture, the cells were collected by centrifugation at 2,000×g and the supernatant was removed. The cells were resuspended in fresh solution containing 50% LB and 50% MS medium at the density of OD₆₀₀=0.5.

TABLE-US-00021 TABLE 21 Fatty acid composition of total leaf lipid from transgenic Nicotiana benthamiana (transient) and Nicotiana tabacum (stable primary transformant) events with the highest EPA levels from each experiment. N. benthamiana N. tabacum 14:0 0.1 0.1 16:0 18.5 17.8 16:1w13t 2.2 3.8 16:1d9 0.1 0 16:3 6.2 5.7 18:0 3.4 3.2 18:1d11 0.3 0.3 20:0 0.5 0.5 22:0 0.2 0.3 24:0 0.1 0.4 18:1 2.9 1.6 18:2ω6 12.6 14.5 Omega-6 18:3ω6 2.3 2.9 20:2ω6 0.0 0.0 20:3ω6 0.1 0.0 20:4ω6 0.3 0.7 Omega-3 18:3ω3 37.1 32.4 18:4ω3 1.6 1.9 20:3ω3 0.1 0.3 20:4ω3 0.3 1.1 20:5ω3 10.7 12.1 22:5ω3 0.3 0.4

[0704] Leaf samples of N. tabacum cultivar W38 grown in vitro were excised and cut into square sections around 0.5-1 cm² in size with a sharp scalpel while immersed in the A. tumefaciens solution. The wounded N. tabacum leaf pieces submerged in A. tumefaciens were allowed to stand at room temperature for 10 minutes prior to being blotted dry on a sterile filter paper and transferred onto MS plates without supplement. Following a co-cultivation period of two days at 24° C., the explants were washed three times with sterile, liquid MS medium, then blotted dry with sterile filter paper and placed on the selective MS agar supplemented with 1.0 mg/L benzylaminopurine (BAP), 0.25 mg/L indoleacetic acid (IAA), 50 mg/L kanamycin and 250 mg/L cefotaxime. The plates were incubated at 24° C. for two weeks to allow for shoot development from the transformed N. tabacum leaf pieces.

[0705] To establish rooted transgenic plants in vitro, healthy green shoots were cut off and transferred into 200 mL tissue culture pots containing MS agar medium supplemented with 25 μg/L IAA, 50 mg/L kanamycin and 250 mg/L cefotaxime. Transgenic shoots were transferred to soil after rooting and grown to maturity in the glasshouse. Sufficiently large leaf discs were taken from 21 mature transgenic plants from and analysed for fatty acid profile as described in Example 1. All transgenic samples were found to contain EPA (Table 21) with the highest level of EPA in a hemizygous primary transformant found to be 12.1% of total leaf lipids. the leaf samples also contained a small amount (<0.5%) of DPA in their lipid, which resulted from elongation of the EPA by a low level of Δ5-elongation activity of the Δ6-elongase. The total ω3 FA (including ALA) to ω6 FA (including LA) ratio was found to be 2.7. Overall conversion efficiencies were calculated to be: OA to EPA=18.4%, LA to EPA=18.9%, ALA to EPA=25.9%. The production of 12.1% EPA is notable especially since the events were hemizygous primary transformants. The ALA to EPA efficiency in particular is close to that observed in stable seed transformants. It is worth noting that the construct did not contain a Δ12 or Δ15-desaturase to increase the conversion of OA and LA to ALA. Increased efficiencies would be expected with addition of these activities.

[0706] Seed from hemizygous transformants is being harvested and sown out to generate homozygous plants.

[0707] Seed set in the top EPA lines appeared normal and seed from lines #10 and #17 germinated well to establish the T₂ generation. The ratio of EPA to null (no EPA) lines indicated that event #28 was single-locus and the T₃ generation of this line was therefore also established. Fatty acid profile analysis of the T₃ population indicated that the transgenes were homozygous with no null events found and a stable amount of EPA. The average amount of EPA in the total leaf lipids in the entire T₃ population was found to be 9.4%±0.3 (Table 22).

TABLE-US-00022 TABLE 22 Representative fatty acid profiles of total leaf lipids from wildtype (WT) and independent transgenic or transiently-transformed lines (EPA). Species are Nicotiana benthamiana (transient transformation), N. tabacum (a stably transformed T₃ population), Vigna unguiculata (stably transformed T₁ event). The errors denote standard deviation of multiple samples. Apparent conversion efficiencies shown at the bottom describe the ω3 pathway and are calculated as the sum of product FAs/sum of substrate + product FAs. N. benthamiana N. tabacum V. unguiculata WT EPA WT EPA WT EPA 16:0 17.7 ± 0.1 18.7 ± 0.2 15.0 ± 0.6 16.5 ± 0.5 18.0 18.2 ± 0.2 16:1ω13t 3.2 ± 0.1 2.2 ± 0 3.5 ± 0.1 3.0 ± 0.3 3.8 2.0 ± 0.9 16:3 6.8 ± 0.1 6.2 ± 0.1 5.2 ± 0.5 5.4 ± 0.3 -- -- 18:0 3.1 ± 0 3.5 ± 0.3 2.2 ± 0.2 2.6 ± 0.1 1.8 4.5 ± 0.4 Minor 1.4 ± 0 1.4 ± 0.1 3.1 ± 0.4 2.5 ± 0.3 2.3 2.5 ± 0.4 OA 1.7 ± 0.1 2.7 ± 0.2 1.6 ± 0.3 2.1 ± 0.3 2.0 4.3 ± 1.3 LA 12.5 ± 0.4 12.7 ± 0.2 17.0 ± 1.1 18.0 ± 0.9 13.4 18.2 ± 3.0 ALA 53.3 ± 0.2 37.2 ± 0.2 52.2 ± 1.9 34.0 ± 0.6 58.6 38.2 ± 0 Omega-6 GLA -- 2.3 ± 0.1 -- 2.3 ± 0.3 -- 0.6 ± 0.2 20:2ω6 0.1 ± 0 -- 0.1 ± 0 0.1 ± 0 -- 0.1 ± 0 DGLA 0.1 ± 0 0.1 ± 0 -- -- -- -- ARA -- 0.3 ± 0 -- 0.7 ± 0.1 -- 0.2 ± 0 Omega-3 SDA -- 1.5 ± 0.1 -- 1.6 ± 0.1 -- 1.5 ± 0 20:3ω3 0.1 ± 0 0.1 ± 0 0.1 ± 0 0.3 ± 0 0.1 ± 0 1.5 ± 0.1 ETA -- 0.4 ± 0 -- 1.1 ± 0.1 -- 0.3 ± 0.2 EPA -- 10.2 ± 0.5 -- 9.4 ± 0.3 -- 7.1 ± 0.2 DPA -- 0.3 ± 0.1 -- 0.4 ± 0 -- 0.8 ± 0.1 Omega-3 Δ6-des 25% 27% 20% conversion Δ6-elo 88% 87% 85% Δ5-des 97% 90% 96% Δ5-elo 3% 4% 10%

[0708] Leaf samples of homozygous T₃ N. tabacum plants were subjected to further biochemical analysis. Total lipids were extracted from freeze-dried leaf material and fractionated by thin-layer chromatography (TLC). EPA was found to be present in N. tabacum TAG at up to 30.1% as well as in the polar lipids at 6.3% (Table 23). It was interesting to note that the EPA produced by the transgenic pathway was present in all of the lipid fractions assessed including TAG, MGDG, DGDG, SQDG, PG, PC, PE, PI and PS. All lipid pools contained low levels of novel intermediate or ω6 LC-PUFA fatty acids with the TAG ratio of novel ω3 to ω6 fatty acids being 10:1.

Stable Transformation of Cowpea

[0709] The chimeric vector pOREO4+11ABGBEC-Cowpea-EPA-insert was transformed into cowpea (Vigna unguiculata) as follows. Mature dry seeds are the preferred starting material although seeds harvested from immature pods at maximum fresh weight of seeds can also be used. Dry seeds are threshed by hand to avoid cracking of seed coats and thus reduce contamination with microorganisms.

[0710] Dry seeds or immature pods are submerged in 70% ethanol for 2 min and then treated for 30 min in 20% commercial bleach (8.4 g/L sodium hypochlorite final concentration). The seeds are then washed several times with sterile water. Immature seeds are removed aseptically from pods while mature seeds are imbibed overnight. Two different explants can be used for multiple shoot production, ie the embryonic axis and the cotyledon itself, preferably the cotyledon with the bisected embryonic axis attached. The shoot and root tips are removed from the axis before wounding at the cotyledonary node, i.e. the point of attachment of the axis to the cotyledon. From an initial comparison of 19 cultivars and lines, it is now clear that most lines of cowpea can be transformed, the only caveat being that different tissue culture conditions need to be optimised for each line.

TABLE-US-00023 TABLE 23 Analysis of young and mature (young|mature) leaf lipid fractions triacylglycerol (TAG), total polar lipid (PL), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) from transgenic Nicotiana tabacum leaf samples. The errors denote standard deviation of multiple samples. Up to 30% EPA was observed in leaf TAG with EPA also distributed throughout the polar lipids. Differences between yound and mature leaf profiles were also observed for several fatty acids. Chloroplastidic Extra-chloroplastidic TAG PL MGDG DGDG SQDG PG PC PE PI PS 16:0 9.8|18.3 17.8|23.8 3.1|3.2 18.0|16.8 48.3|50.0 21.0|26.4 22.9|30.0 24.0|30.5 38.7|43.3 31.9|36.2 16:1ω13t 0|0 3.4|3.1 0|0 0|0 0|0 34.0|32.0 0|0 0|0 0|0 1.0|1.4 16:3 0.2|0.9 5.6|6.4 14.8|19.4 1.2|1.8 0.4|1.2 0|0 0|0 0|0 0|0 0|0 18:0 7.3|3.7 2.9|3.9 1.1|1.2 3.5|3.5 5.4|7.1 4.7|6.9 6.6|9.1 11.0|11.4 9.4|9.3 20.2|19.4 Minor 2.5|2.9 1.4|2.4 1.0|0.4 0.8|1.0 1.9|2.1 1.0|1.5 1.4|1.6 4.9|4.1 6.5|7.7 2.5|3.7 OA 5.5|0.8 2.8|1.1 0.8|0.3 1.8|1.0 2.7|1.3 5.3|4.9 8.1|2.9 2.5|1.1 2.5|0.8 4.9|2.3 LA 27.7|13.7 17.3|12.3 8.0|6.8 9.2|10.5 11.7|8.9 17.1|13.2 39.2|25.2 37.9|28.5 22.0|13.4 24.4|17.1 ALA 9.6|17.2 39.0|34.4 60.3|51.9 61.2|58.6 23.7|21.5 15.7|14.1 7.3|18.2 5.5|10.5 7.6|10.0 4.8|10.5 Omega-6 GLA 2.5|3.0 1.5|2.1 2.1|3.0 1.1|1.8 1.4|1.9 0.2|0 1.8|2.5 1.7|2.7 0.8|0.9 1.1|1.3 20:2ω6 0|0 0.1|1.1 0|0 0|0 0|0 0|0 0|0 0.5|0 0|0 0|0 DGLA 0|0 0|0 0|0 0|0 0|0 0|0 0|0 0|0 0|0 0|0 ARA 0.6|0.9 0.1|0.2 0.2|0.4 0|0 0|0 0|0 0.3|0.3 0.4|0.4 0.4|0.6 0|0.2 Omega-3 SDA 4.0|7.6 1.6|2.0 1.7|2.0 0.6|0.7 1.2|1.2 0|0 2.1|3.6 1.3|2.0 0.8|0.8 0.9|1.6 20:3ω3 0.2|0.3 0.1|0.2 0|0 0.2|0.3 0|0 0|0.1 0.2|0 0.3|0.4 0|0 0|0 ETA 0.9|0.2 0.2|0.3 0|0.2 0|0.3 0|0 0|0 0.2|0 0.4|0.2 0.1|0.2 0|0 EPA 28.8|30.1 6.1|6.3 6.9|11.2 2.3|3.6 3.4|4.6 1.0|0.8 9.7|6.4 9.1|7.8 11.2|12.8 8.4|6.2 DPA 0.4|0.5 0|0 0|0.1 0|0 0|0 0|0 0.3|0.4 0.5|0.4 0|0.2 0|0.1

[0711] The selectable marker genes, bar or NptII can be used for transfomation. The Agrobacterium tumefaciens strain AGL1 is the preferred strain for cowpea transformation. Agrobacterium containing the pORE04+11ABGBEC-Cowpea-EPA-insert vector is cultured overnight at 28° C. on a shaker at 180 rpm and the suspension is centrifuged at 8000 g for 10 min and re-suspended in Medium 1 (MS-basic medium diluted one in ten and containing 30 g/l sucrose, 20 mM 2-MES, adjusted to pH 5.6 prior to autoclaving, supplemented with filter sterilized MS-vitamins, 100 mg/l myo-inositol, 1.7 mg/l BAP, 0.25 mg/l GA₃, 0.2 mM acetosyringone, 250 mg/l Na-thiosulphate, 150 mg/l dithiothreitol and 0.4 g/l L-cysteine). The explants are submerged without shaking in the bacterial suspension for one hour following wounding in the meristematic regions with a scalpel. The treated explants are then blotted on sterile filter paper and transferred to solidified Medium 2 (Medium I containing 0.8% agar) overlayed with filter paper. After four days of co-cultivation, explants are transferred to Medium 3 (full strength MS medium, supplemented with 100 mg/l myo-inositol, 150 mg/l timentin, 30 g/L sucrose, 3 mM MES, 1.7 mg/L BAP, 5 mg/L PPT or 25-50 mg/L geneticin or 150 mg/L kanamycin, 0.8 g/L agar and adjusted to pH 5.6) for shoot initiation and selection of transformed shoots. After two weeks the first shoots are visible. The cotyledons are removed from the cotyledonary node region and cultures are transferred to fresh Medium 3. Cultures are transferred to fresh Medium 3 every two weeks following removal of dead and dying tissue. The first four subcultures are on kanamycin selection followed by alternating with geneticin and kanamycin. After six sub-cultures, the surviving green shoots are transferred to Medium 4 (Medium 3 without BAP but supplemented with 0.5 mg/l GA₃, 50 mg/l asparagine, 0.1 mg/l 3-indoleacetic acid (IAA), 150 mg/l timentin, and either PPT (10 mg/l), geneticin (50 mg/L) or kanamycin (150 mg/L), for shoot elongation. The shoots are sub-cultured every two weeks until single shoots are more than 1 cm long. These larger shoots are transferred from petri dishes to culture jars (80 mm height) for further growth under selection.

[0712] The majority of the regenerated shoots can be rooted in vitro, and the rooted plants are transferred to soil and allowed to establish in a high humidity chamber for 14-21 days before transfer to ambient greenhouse conditions.

[0713] To enhance gene transfer to cowpea, co-culture media is supplemented with thiol compounds. The addition of L-cysteine, dithiothreitol, and sodium thiosulfate reduces browning of wounded tissue.

[0714] Large numbers of cowpea explants can be processed in a simplified protocol. In brief, the protocol consists of the following steps: imbibition of sterilized mature seeds overnight in water, explants are derived by longitudinally bisecting the seed as a result of which, the split embryonic axis (with shoot and root apices removed) is still attached to the cotyledon, infection with Agrobacterium strain AGL1 aided by local wounding in the meristematic regions, co-culture on medium containing thiol compounds over 4 days at 25° C. in light, shoot initiation and elongation on medium containing selective agents, shoots are rooted in vitro and transferred to greenhouse conditions for flowering and seed setting, PCR or enzyme analysis of putative transgenic plants, and screening of next generation progeny by PCR or enzyme activity.

[0715] The progeny of transgenic T₀ plants are normal in phenotype. The transgenes are transmitted to the progeny and homozygous T₂ plants are identified by screening their T₃ progeny for enzyme activity or by PCR.

[0716] Using this transformation system about 10 transgenic plants are produced per 1000 explants, which is similar to the transformation frequency for other legumes. Depending on the cultivar or line to be transformed, this protocol requires 5-8 months from explant preparation to harvested T₁ seeds.

[0717] The transformation system is used to introduce the pORE04+11ABGBEC-Cowpea-EPA-insert binary vector into regenerated, transformed cowpea plants.

[0718] Modifications to the pOREO4+11ABGBEC-Cowpea-EPA-insert binary vector are made in which genes encoding a Δ5-elongase and Δ4-desaturase are added, to provide a genetic construct which confers the ability to further convert the produced EPA to DHA. The construct is transformed into plants for production of DHA in vegetative tissues.

[0719] EPA was found to be present in the small number of events surviving chemical selection. The highest line contained 7.1%±0.2 EPA in the total leaf lipids. The rate of transformation was lower than usually experienced for cowpea with only six lines confirmed transgenic. It is, as yet, unknown what caused this effect although it is interesting to note that a larger than usual proportion of transgenic events contained incomplete T-DNA regions. It is possible that the large construct size contributed to the reduced efficiency. The apparent conversion efficiencies of each of the three transgenic enzymes were also calculated (Table 22). Results were broadly similar in all three species with good conversion to EPA after initial Δ6-desaturation of the native ALA. Some Δ5-elongation of EPA to DPA was noted despite the absence of a specific Δ5-elongase. The P. cordata Δ6-elongase has previously been shown to have a low level of Δ9-elongase activity (i.e. 18:3.sup.Δ9, 12, 15 to 20:3.sup.Δ11, 14, 17 conversion) although no Δ5-elongase activity was detected in a yeast assay.

Example 10

Testing Variations of Δ12-Desaturase Genes

Binary Vector Construction

[0720] In an attempt to test and compare a series of chimeric Δ12-desaturase genes, several binary vectors were made which were used to transform A. thaliana and B. napus. The binary vectors pJP3365, pJP3366, pJP3367, pJP3368 and pJP3369 each contained genes that encoded the P. pastoris ω3-desaturase (SEQ ID NO:12) and M. pusilla Δ6-desaturase (SEQ ID NO:16) enzymes, and one of a series of Δ12-desaturases. The Δ12-desaturases were from Cryptococcus neoformans (Accession No. XP_--570226 in pJP3365), a version of the Cryptococcus neoformans Δ12-desaturase which contained a L151M mutation in an attempt to increase gene activity (in pJP3366), Lachancea kluyveri (SEQ ID NO:10 in pJP3367), Synechocystis PCC6803 (Accession No. BAA18169 in pJP3368) and Crepis palaestina (Accession No. CAA76157, Lee et al., 1998, in pJP3369). The Crepis desaturase was the only plant desaturase in the series; the others were fungal enzymes. The vectors were made by inserting a plant codon-optimised protein coding region, except for the Crepis palestina Δ12-desaturase which was wildtype, for each Δ12-desaturase into the NotI site of the vector pJP3364 (see FIG. 12), in the orientation operably linked to the FP1 promoter to provide for seed-specific expression of each desaturase. The vector pJP3364 already contained the chimeric genes encoding the P. pastoris ω3-desaturase and M. pusilla Δ6-desaturase, each under the control of seed-specific promoters (FIG. 12). The combination of the three fatty acid biosynthesis enzymes, namely Δ12-desaturase, ω3-desaturase and Δ6-desaturase, was designed to assemble a pathway to convert oleic acid (18:1.sup.Δ9) to SDA (18:4.sup.Δ6, 9, 12, 15). Assays were therefore carried out to measure the level of SDA production in transformed seeds.

A. thaliana and B. napus Transformation and Analysis

[0721] The chimeric binary vectors were introduced into A. tumefaciens strain AGL1 and cells from cultures of the transformed Agrobacterium used to transform fad2 mutant A. thaliana plants using the floral dip method for transformation (Clough and Bent, 1998). After maturation, the T₁ seeds from the treated plants were harvested and plated on MS plates containing kanamycin for selection of plantlets having the NptII selectable marker gene present on the T-DNA of each chimeric vector. Surviving T₁ seedlings were transferred to soil. After allowing the plants to self-fertilise and growing them to maturity, the T₂ seeds from these plants were harvested and the fatty acid composition of seed lipids analysed by GC.

[0722] The chimeric vector pJP3367 was also used to transform B. napus by the method described in Example 4 to generate 12 transgenic events. SDA was found to range from 0.6% to 2.2% in pooled seed of the plants, and nine individual seeds from the transgenic plant with the highest SDA transgenic plant were analysed for fatty acid composition. Fatty acid composition data from such analysis is shown in Table 24.

[0723] The data showed that the Δ12-desaturase activity expressed from each of the T-DNAs in both A. thaliana and B. napus were unexpectedly low, providing an enzyme conversion efficiency of about 20% rather than the 70-80% seen with the same expression cassette in the GA7 construct (Examples 2 and 3). The reason for this relatively poor expression of the Δ12-desaturase genes from these vectors is unclear but could be related to the position of the genes in the construct as a whole.

[0724] In contrast, RT-PCR expression analysis demonstrated that the P. pastoris ω3-desaturase and M. pusilla Δ6-desaturase genes on the T-DNAs were relatively well expressed in the transformed seed. Table 24 includes the Δ6-desaturase conversion efficiencies in the transformed seeds, which ranged from about 11% to about 25% in the one B. napus transformed line. This was considerably higher than the Δ6-desaturase conversion efficiency of about 7% seen in the B. napus seeds transformed with the GA7 construct (Example 4).

TABLE-US-00024 TABLE 24 Fatty acid composition as a percentage of total fatty acids in seed oil from single seeds from a T₁ Brassica napus plant transformed with the T-DNA from pJP3367. SDA (18:4ω3) is shown in bold. CT110- CT110- CT110- CT110- CT110- CT110- CT110- CT110- CT110- Sample 3#1 3#2 3#3 3#4 3#5 3#6 3#7 3#8 3#9 C14:0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 C16:0 4.3 4.2 4.1 4.5 3.8 4.3 4.0 5.0 4.7 16:1d7 0.1 0.1 0.1 0.1 0.0 0.1 0.1 0.1 0.1 C16:1d9 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.3 16:3 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 C18:0 1.9 1.9 1.3 1.8 2.1 1.8 2.4 3.1 2.2 C18:1 58.1 59.4 55.5 59.1 62.1 56.0 57.2 52.0 53.2 C18:1d11 3.5 3.6 3.0 3.2 2.9 3.6 3.2 4.4 3.5 C18:2 18.4 17.1 19.2 17.3 17.4 18.7 19.0 20.3 20.2 C18:3ω6 0.3 0.2 0.3 0.2 0.2 0.2 0.2 0.2 0.3 C18:3ω3 8.2 9.0 11.1 8.6 7.5 10.2 9.8 9.3 9.8 C20:0 0.5 0.5 0.4 0.5 0.6 0.5 0.6 0.7 0.6 18:4ω3 2.4 2.0 2.8 2.5 1.4 2.6 1.3 2.4 3.2 C20:1d11 1.1 1.1 1.2 1.2 1.2 1.1 1.2 1.1 1.1 20:1iso 0.03 0.03 0.03 0.03 0.01 0.03 0.02 0.03 0.02 C20:2ω6 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 C22:0 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.2 C24:0 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.3 0.2 C24:1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Δ6-des % 22.9 17.9 20.3 22.8 15.8 20.2 11.7 20.9 24.9

[0725] Therefore, to take advantage of the higher Δ6-desaturase conversion efficiencies conferred by the T-DNA from pJP3367, B. napus plants transformed with this T-DNA were crossed to plants transformed with the T-DNA from pJP3416-GA7 (Example 4) to produce progeny plants and seeds carrying both T-DNAs. The fatty acid composition of oil extracted from F seeds is analysed by GC for DHA content and other fatty acid contents. Increased DHA levels are observed as a consequence of increased expression of the Δ6-desaturase. Plants which are homozygous for both T-DNAs are produced and should produce higher levels of DHA.

Example 11

Increasing Accumulation of Fatty Acids by Using Silencing Suppressor Proteins

Binary Vector Construction

[0726] WO 2010/057246 describes the use of silencing suppressor proteins (SSP) to increase transgene expression in the seeds of plants. To demonstrate that the use of such proteins could enhance and stabilise the production of LC-PUFA in oilseeds over several generations, several SSP were selected for testing, namely V2 (Accession No. GU178820.1), p19 (Accession No. AJ288943.1), p38 (Accession No. DQ286869.1) and P0^PE (Accession No. L04573.1). p19 is a suppressor protein from Tomato Bushy Stunt Virus (TBSV) which binds to 21 nucleotide long siRNAs before they guide Argonaute-guided cleavage of homologous RNA (Voinnet et al., 2003). V2, a suppressor protein from Tomato Yellow Leaf Curl Virus (TYLCV), binds to the plant protein SGS3 (Glick et al., 2008), a protein thought to be required for the production of double stranded RNA intermediates from ssRNA substrates (Beclin et al., 2002), or binds to dsRNA structures that have a 5' overhangs (Fukunaga et al., 2009). p38 is a suppressor protein from Turnip Crinkle Virus (TCV) which interferes with plant silencing mechanisms by binding to Dicer and Argonaute proteins (Azevedo et al., 2010). P0 proteins such as P0^PE and RPV-P0, from poleroviruses, target Argonaut proteins for enhanced degradation (Baumberger et al., 2007; Bortolamiol et al., 2007, Fusaro et al., 2012). Genetic constructs were therefore prepared for expression of these SSP in plant seed in combination with a set of fatty acid biosynthesis genes for production of ARA (20:4.sup.Δ5, 8, 11, 14) from LA (18:1.sup.Δ9, 12), as follows.

[0727] The fatty acid biosynthesis genes encoding the Isochrysis galbana Δ9-elongase and the Pavlova salina Δ8- and Δ5-desaturases and the bacterial selection marker were obtained on a single DNA fragment from pJP3010 by digestion with PmeI and AvrII giving rise to a 9560 bp fragment. The Δ9-elongase coding region on this fragment was joined to an A. thaliana FAE1 promoter (pAtFAE1) and a conlinin transcription termination/polyadenylation region (LuCnl2-3'). The desaturase coding regions were each joined to a truncated napin FP1 promoter (pBnFP1) and a nos3' transcription termination/polyadenylation region. The three fatty acid biosynthesis genes on this fragment were oriented and spaced in the same manner as in pJP107 (Petrie et al., 2012) and encoded the same proteins as pJP107. The DNA fragment also comprised a pFP1:GFiP:nos3' gene from pCW141 (see WO02010/057246) which encoded a green fluorescent protein (GFP). This screenable marker gene was used as a visual seed-specific marker, allowing simple and non-destructive identification and thereby selection of transgenic seed comprising and expressing the gene.

[0728] The PmeI-AvrII fragment was inserted into the PmeI-AvrII site of each of a series of five vectors, each containing a different SSP gene (WO2010/057246), resulting in the genetic constructs designated pFN045, pFN046, pFN047, pFN048 and pFN049. These comprise the genes encoding the SSPs P0^PE, p38, p19, 35S:V2 and V2, respectively. Each of the SSP genes was under the control of the FP1 promoter and ocs3' transcription termination/polyadenylation region except in the construct pFN048 where the V2 coding region was under the control of the constitutive CaMV 35S promoter. The SSP gene in each case was within the T-DNA region of the constructs, adjacent to the right border (RB) of the T-DNA. A sixth construct, pFN050 which lacked any SSP coding sequence, was made by digesting pFN045 with AhdI and NheI followed by recircularisation with DNA ligase to delete the FP1:P0^PE gene. Each of the six constructs comprised an NptII selectable marker gene within the T-DNA and adjacent to the left border of the T-DNA. All of the constructs had an RK2 origin of replication for maintenance of the plasmids in Agrobacterium.

Transformation of A. thaliana with ARA Expression Vectors in Combination with SSPs

[0729] To transform the genotype MC49 of Arabidopsis, which is a fad2/faeI double mutant with high linoleic acid levels in its seed lipid, plants were treated by the floral dip method (Clough and Bent, 1998) with A. tumefaciens strain GV3101 separately transformed with each of the six constructs pFN045-pFN050. The treated plants were grown to maturity and T₁ seeds harvested from them were plated on MS media containing kanamycin to select for transformed T₁ plants. Screening for GFP expression in the seed was also used as a visual marker for transformed T₁ seeds. The seedlings which survived on MS/Kan plates or which were obtained from GFP-positive seeds were transferred to soil and grown to maturity for T₂ seeds. The numbers of transformed plants obtained were 5, 14, 32, 8, 23 and 24 for the transformations with pFN045, pFN046, pFN047, pFN048, pFN049 and pFN050, respectively. It was discovered at this stage that the gene encoding p38 in pFN046 was not functional and therefore plants transformed with the vector pFN046 were considered as additional controls i.e. essentially the same as for pFN050.

[0730] About 100 pooled T₂ seeds were taken from each transformed plant for fatty acid composition determination of seed lipid by FAME preparation and GC analysis. Six T₂ seedlings from each transgenic line were also grown to produce T₃ seeds.

[0731] The fatty acid composition in total lipid extracted from the T₂ seeds was determined using GC. The analysis showed a range of levels of ARA and the intermediates EDA (20:2ω6) and DGLA (20:3ω6) in the T₂ populations. The data for ARA is shown in FIGS. 13 and 14.

[0732] FIG. 13 shows a box-plot analysis of the ARA level in the lipid of the populations of the T₂ seeds. It was evident that the median (50^th percentile) level of ARA in the populations of seeds which contained the FP1:p19 and 35S:V2 genes in addition to the ARA biosynthetic genes was significantly higher than in seeds containing the defective FP1:p38 gene or the control T-DNA from pFP050 which did not contain an SSP gene. The average ARA levels for seeds transformed with genes encoding p19 and V2 were greater than for seeds transformed with the p38 gene or those without an SSP (FIG. 14). One FP1:p19 and two FP1:V2 lines exhibited about 19%, 20% and 23% ARA, respectively. These were outliers and therefore not included in the calculations for the box-plot analysis. Fewer plants transformed with the T-DNAs comprising the genes FP1:P0^PE and 35S:V2 survived compared to the other constructs; it is thought that these genes could be detrimental to plant health in the MC49 background.

[0733] Not only were the ARA levels significantly different among the constructs, the levels in seed lipid of the first intermediate of the pathway from LA to ARA, namely EDA (20:2ω6), was observed to be lower in lines expressing either V2 or p19 than in seeds lacking an SSP or containing the p38 construct (FIG. 15). In T₃ seeds, one population containing the construct expressing p19 exhibited 38% ARA as a percentage of total fatty acids in the seed lipid.

[0734] A range of transgenic T₃ lines were progressed to the T₄ generation. The levels of ARA in the T₄ seeds expressing V2 were either the same as compared to the previous generation, or indeed exhibited increased levels compared to their T3 parents (FIG. 16). The lines expressing p19 showed more varied ARA levels. The ARA level was decreased in some lines while in others it was the same or increased compared to the T3 parents. In contrast, the lines containing the defective p38 gene or lacking an SSP generally showed a decline in the level of ARA and an increase in the levels of intermediates (FIG. 18). In some of these lines, ARA was reduced to about 1% and levels of EDA had increased to about 20%. The mean levels of ARA in T₄ seeds were higher for lines expressing p19 and V2 compared to lines expressing p38 or lacking an SSP (FIG. 17).

[0735] This experiment showed that the expression of an SSP in seeds of a transgenic plant along with additional genes for a LC-PUFA biosynthetic pathway not only increased the level of production of the desired fatty acid in the first generation of progeny, but also stabilised the level of the fatty acid production in later generations such as the third or fourth generation of progeny. The increased fatty acid production was accompanied by decreased levels of intermediate fatty acids in the biosynthetic pathway. The SSP's p19 and V2 expressed from seed-specific promoters were preferred. The construct designed to express the p38 SSP was defective and no useful data were obtained with this construct. The V2 SSP and its homologs from other viruses are thought to be particularly preferred because they allow maximal expression of the biosynthetic pathway genes and the simultaneous silencing of other genes in the same cells in the developing seed.

Example 12

Assaying Sterol Content and Composition in Oils

[0736] The phytosterols from 12 vegetable oil samples purchased from commercial sources in Australia were characterised by GC and GC-MS analysis as O-trimethylsilyl ether (OTMSi-ether) derivatives as described in Example 1. Sterols were identified by retention data, interpretation of mass spectra and comparison with literature and laboratory standard mass spectral data. The sterols were quantified by use of a 5β(H)-Cholan-24-ol internal standard. The basic phytosterol structure and the chemical structures of some of the identified sterols are shown in FIG. 19 and Table 25.

[0737] The vegetable oils analysed were from: sesame (Sesamum indicum), olive (Olea europaea), sunflower (Helianthus annus), castor (Ricinus communis), canola (Brassica napus), safflower (Carthamus tinctorius), peanut (Arachis hypogaea), flax (Linum usitatissimum) and soybean (Glycine max). In decreasing relative abundance, across all of the oil samples, the major phytosterols were: β-sitosterol (range 28-55% of total sterol content), Δ5-avenasterol (isofucosterol) (3-24%), campesterol (2-33%), Δ5-stigmasterol (0.7-18%), Δ7-stigmasterol (1-18%) and Δ7-avenasterol (0.1-5%). Several other minor sterols were identified, these were: cholesterol, brassicasterol, chalinasterol, campestanol and eburicol. Four C29:2 and two C30:2 sterols were also detected, but further research is required to complete identification of these minor components. In addition, several other unidentified sterols were present in some of the oils but due to their very low abundance, the mass spectra were not intense enough to enable identification of their structures.

[0738] The sterol contents expressed as mg/g of oil in decreasing amount were: canola oil (6.8 mg/g), sesame oil (5.8 mg/g), flax oil (4.8-5.2 mg/g), sunflower oil (3.7-4.1 mg/g), peanut oil (3.2 mg/g), safflower oil (3.0 mg/g), soybean oil (3.0 mg/g), olive oil (2.4 mg/g), castor oil (1.9 mg/g). The % sterol compositions and total sterol content are presented in Table 26.

TABLE-US-00025 TABLE 25 IUPAC/systematic names of identified sterols. Sterol No. Common name(s) IUPAC/Systematic name 1 cholesterol cholest-5-en-3β-ol 2 brassicasterol 24-methylcholesta-5,22E-dien-3β-ol 3 chalinasterol/24-methylene 24-methylcholesta-5,24(28)E-dien- cholesterol 3β-ol 4 campesterol/24- 24-methylcholest-5-en-3β-ol methylcholesterol 5 campestanol/24- 24-methylcholestan-3β-ol methylcholestanol 7 Δ5-stigmasterol 24-ethylcholesta-5,22E-dien-3β-ol 9 ergost-7-en-3β-ol 24-methylcholest-7-en-3β-ol 11 eburicol 4,4,14-trimthylergosta-8,24(28)-dien- 3β-ol 12 β-sitosterol/24- 24-ethylcholest-5-en-3β-ol ethylcholesterol 13 D5-avenasterol/ 24-ethylcholesta-5,24(28)Z-dien-3/β- isofucosterol ol 19 D7-stigmasterol/stimast-7- 24-ethylcholest-7-en-3/β-ol en-3b-ol 20 D7-avenasterol 24-ethylcholesta 7,24(28)-dien-3β-ol

[0739] Among all the seed oil samples, the major phytosterol was generally β-sitosterol (range 30-57% of total sterol content). There was a wide range amongst the oils in the proportions of the other major sterols: campesterol (2-17%), Δ5-stigmasterol (0.7-18%), Δ5-avenasterol (4-23%), Δ7-stigmasterol (1-18%). Oils from different species had a different sterol profile with some having quite distinctive profiles. In the case of canola oil, it had the highest proportion of campesterol (33.6%), while the other species samples generally had lower levels, e.g. up to 17% in peanut oil. Safflower oil had a relatively high proportion of Δ7-stigmasterol (18%), while this sterol was usually low in the other species oils, up to 9% in sunflower oil. Because they were distinctive for each species, sterol profiles can therefore be used to help in the identification of specific vegetable or plant oils and to check their genuineness or adulteration with other oils.

TABLE-US-00026 TABLE 26 Sterol content and composition of assayed plant oils. Sun- Saf- flower flower Sterol Sterol common Sun- cold- Saf- cold- Flax Flax number* name Sesame Olive flower pressed Castor Canola flower pressed Peanut (linseed) (linseed) Soybean 1 cholesterol 0.2 0.8 0.2 0.0 0.1 0.3 0.2 0.1 0.2 0.4 0.4 0.2 2 brassicasterol 0.1 0.0 0.0 0.0 0.3 0.1 0.0 0.0 0.0 0.2 0.2 0.0 3 chalinasterol/24- 1.5 0.1 0.3 0.1 1.1 2.4 0.2 0.1 0.9 1.5 1.4 0.8 methylene cholesterol 4 campesterol/24- 16.2 2.4 7.4 7.9 8.4 33.6 12.1 8.5 17.4 15.7 14.4 16.9 methylcholesterol 5 campestanol/24- 0.7 0.3 0.3 0.1 0.9 0.2 0.8 0.8 0.3 0.2 0.2 0.7 methylcholestanol 6 C29:2* 0.0 0.0 0.1 0.2 0.0 0.1 0.5 0.5 0.0 1.2 1.3 0.1 7 Δ5-stigmasterol 6.4 1.2 7.4 7.2 18.6 0.7 7.0 4.6 6.9 5.1 5.8 17.6 8 unknown 0.5 1.3 0.7 0.6 0.8 0.7 0.7 1.3 0.4 0.7 0.6 1.3 9 ergost-7-en-3β-ol 0.1 0.1 1.9 1.8 0.2 0.4 2.7 4.0 1.4 1.4 1.4 1.0 10 unknown 0.0 1.3 0.9 0.8 1.2 0.9 1.8 0.7 1.2 0.7 0.5 0.7 11 eburicol 1.6 1.8 4.1 4.4 1.5 1.0 1.9 2.9 1.2 3.5 3.3 0.9 12 β-sitosterol/24- 55.3 45.6 43.9 43.6 37.7 50.8 40.2 35.1 57.2 29.9 28.4 40.2 ethylcholesterol 13 Δ5-avenasterol/ 8.6 16.9 7.2 4.1 19.3 4.4 7.3 6.3 5.3 23.0 24.2 3.3 isofucosterol 14 triterpenoid 0.0 2.4 0.9 1.1 0.0 0.0 1.6 1.9 0.0 0.0 0.0 0.9 alcohol 15 triterpenoid 0.0 0.0 0.7 0.6 0.0 0.0 2.8 1.8 0.0 0.0 0.3 0.0 alcohol 16 C29:2* 0.0 0.5 0.7 0.7 1.5 1.2 2.8 1.9 2.0 1.0 0.7 0.5 17 C29:2* 1.0 0.9 2.3 2.4 0.6 0.4 1.3 1.9 0.9 1.0 1.0 1.0 18 C30:2* 0.0 0.0 0.0 0.0 1.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 19 Δ7-stigmasterol/ 2.2 7.1 9.3 10.9 2.3 0.9 10.5 18.3 1.1 7.9 8.7 5.6 stigmast-7-en-3β- ol 20 Δ7-avenasterol 1.3 0.1 4.0 3.6 0.6 0.2 2.0 4.7 0.7 0.4 0.4 0.6 21 unknown 0.7 7.1 0.9 0.8 0.0 0.4 0.3 0.4 0.0 3.0 3.6 0.0 22 unknown 0.3 0.0 0.3 0.9 0.0 0.0 1.2 1.3 0.2 0.1 0.0 0.3 23 unknown 0.2 0.2 0.3 0.3 0.2 0.1 0.3 0.2 0.2 0.1 0.2 0.5 24 unknown 0.0 3.1 0.9 1.3 0.6 0.4 0.2 0.4 0.7 1.7 1.9 0.8 25 unknown 0.9 0.4 0.3 0.5 0.3 0.1 0.5 0.7 0.3 0.1 0.1 0.6 26 C30:2 2.2 6.0 4.6 5.7 1.4 0.6 1.0 1.2 1.2 1.2 1.1 5.2 27 unknown 0.0 0.4 0.4 0.3 0.3 0.2 0.1 0.2 0.3 0.1 0.0 0.3 Sum 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 Total sterol (mg/g 5.8 2.4 4.1 3.7 1.9 6.8 3.2 3.0 3.2 4.8 5.2 3.0 oil) C29:2* and and C30:2* denotes a C29 sterol with two double bonds and a C30 sterol with two double bonds, respectively

[0740] Two samples each of sunflower and safflower were compared, in each case one was produced by cold pressing of seeds and unrefined, while the other was not cold-pressed and refined. Although some differences were observed, the two sources of oils had similar sterol compositions and total sterol contents, suggesting that processing and refining had little effect on these two parameters. The sterol content among the samples varied three-fold and ranged from 1.9 mg/g to 6.8 mg/g. Canola oil had the highest and castor oil the lowest sterol content.

Example 13

Increasing Accumulation of DHA at the Sn-2 TAG Position

[0741] The present inventors considered that DHA accumulation at the sn-2 position in TAG could be increased by co-expressing an 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT) together with the DHA biosynthesis pathway such as conferred by the GA7 construct or its variants. Preferred LPAATs are those which can act on polyunsaturated C22 fatty acyl-CoA as substrate, resulting in increased insertion of the polyunsaturated C22 chain at the sn-2 position of LPA to form PA, relative to the endogenous LPAAT. Cytoplasmic LPAAT enzymes often display varied substrate preferences, particularly where the species synthesises and accumulates unusual fatty acids in TAG. A LPAAT2 from Limnanthes douglasii was shown to use erucoyl-CoA (C22:1-CoA) as a substrate for PA synthesis, in contrast to an LPAAT1 from the same species that could not utilise the C22 substrate (Brown et al., 2002).

[0742] Known LPAATs were considered and a number were selected for testing, including some which were not expected to increase DHA incorporation at the sn-2 position, as controls. The known LPAATs included: Arabidopsis thaliana LPAAT2: (SEQ ID NO: 63, Accession No. ABG48392, Kim et al., 2005), Limnanthes alba LPAAT (SEQ ID NO: 64, Accession No. AAC49185, Lassner et al., 1995), Saccharomyces cerevisiae Slc1p (SEQ ID NO: 65, Accession No. NP_--010231, Zou et al., 1997), Mortierella alpina LPAATI (SEQ ID NO: 67, Accession No. AED33305; U.S. Pat. No. 7,879,591) and Brassica napus LPAATs (SEQ ID NO: 68 and SEQ ID NO:69, Accession Nos ADC97479 and ADC97478 respectively). These were chosen to cover three groups of LPAAT enzymes: 1) control plant seed LPAATs with typically low activity on unusual long-chain polyunsaturated fatty acids (including the Arabidopsis and Brassica LPAATs), 2. LPAATs that had previously been demonstrated to act on C22 fatty acids by using C22 acyl-CoA as substrate, in this case erucic acid C22:1 (including the Limnanthes and Saccharomyces LPAATs), 3. LPAATs which the inventors considered likely to be able to utilise long-chain polyunsaturated fatty acids such as EPA and DHA as substrates (including the Mortierella LPAAT).

[0743] The Arabidopsis LPAAT2 (also designated LPAT2) is an endoplasmic reticulum-localised enzyme shown to have activity on C16 and C18 substrates, however activity on C20 or C22 substrates was not tested (Kim et al., 2005). Limnanthes alba LPAAT2 was demonstrated to insert a C22:1 acyl chain into the sn-2 position of PA, although the ability to use DHA as a substrate was not tested (Lassner et al., 1995). The selected S. cerevisiae LPAAT Slc1p was shown to have activity using 22:1-CoA in addition to 18:1-CoA as substrates, indicating a broad substrate specificity with respect to chain length (Zou et al., 1997). Again, DHA-CoA and other LC-PUFAs were not tested as substrates. The Mortierella LPAAT had previously been shown to have activity on EPA and DHA fatty acid substrates in transgenic Yarrowia lipolytica (U.S. Pat. No. 7,879,591).

[0744] Additional LPAATs were identified by the inventors. Micromonas pusilla is a microalga that produces and accumulates DHA in its oil, although the positional distribution of the DHA on TAG in this species has not been confirmed. The Micromonas pusilla LPAAT (SEQ ID NO: 66, Accession No. XP_--002501997) was identified by searching the Micromonas pusilla genomic sequence using the Arabidopsis LPAAT2 as a BLAST query sequence. Several candidate sequences emerged and the sequence XP_--002501997 was synthesised for testing as a likely LPAAT enzyme with activity on C22 LC-PUFA. The Ricinus communis LPAAT was annotated as a putative LPAAT in the castor genome sequence (Chan et al., 2010). Four candidate LPAATs from the castor genome were synthesised and tested in crude leaf lysates of infiltrated N. benthamiana leaf tissue. The candidate sequence described here showed LPAAT activity.

[0745] A number of candidate LPAATs were aligned with known LPAATs on a phylogenetic tree (FIG. 20). It was noted that the putative Micromonas LPAAT did not cluster with the putative C22 LPAATs but was a divergent sequence.

[0746] As an initial test of various LPAATs for their ability to use DHA-CoA as substrate, chimeric genetic constructs are made for constitutive expression of exogenous LPAATs in N. benthamiana leaves, each under the control of the 35S promoter, as follows: 35S:Arath-LPAAT2 (Arabidopsis ER LPAAT); 35S:Ricco-LPAAT2; 35S:Limal-LPAAT (Limnanthes alba LPAAT); 35S:Sacce-Slc1p (S. cerevisiae LPAAT); 35S:Micpu-LPAAT (Micromonas pusilla LPAAT); 35S:Moral-LPAAT1 (Mortierella alpina LPAAT). A 35S:p19 construct lacking an exogenous LPAAT is used as a control in the experiment. Each of these constructs is introduced via Agrobacterium into N. benthamiana leaves as described in Example 1, and 5 days after infiltration, the treated leaf zones are excised and ground to make leaf lysates. Each lysate includes the exogenous LPAAT as well as the endogenous enzymes for synthesizing LPA. In vitro reactions are set up by separately adding ¹⁴C-labelled-OA, -LA or -ALA (C18 substrates), -ARA (a C20 substrate) and -DHA (C22) to the lysates, in triplicate. Reactions are incubated at 25° C. and the level of incorporation of the ¹⁴C labelled fatty acids into PA determined by TLC. The ability of each LPAAT to use DHA relative to ARA and the C18 fatty acids is calculated. The meadowfoam, Mortierella and Saccharomyces LPAATs were found to have activity on DHA substrate, with radiolabelled PA appearing for these but not the other LPAATs. All LPAATs were confirmed active by a similar oleic acid feed.

[0747] To test LPAAT activity in seeds, several of the protein coding sequences or LPAATs are inserted into a binary vector under the control of a conlinin (pLuCnl1) promoter. The resultant genetic constructs containing the chimeric genes, Cnl1:Arath-LPAAT (negative control), Cnl1:Limal-LPAAT, Cnl:Sacce-Slc1p, and Cnl1:Moral-LPAAT, respectively, are then used transform B. napus and A. thaliana plants to generate stable transformants expressing the LPAATs in a seed-specific manner. The transformed plants having the Cnl1:LPAAT constructs are crossed with plants expressing the GA7 construct or its variants (Example 5) which produce DHA in the seed to result in increased incorporation of DHA at the sn-2 position of TAG. The constructs are also used to transform B. napus, C. sativa and A. thaliana plants that already contain the GA7 construct and variants thereof (Examples 2 to 5) to generate progeny carrying both the parental and LPAAT genetic constructs. Increased incorporation of DHA at the sn-2 position of TAG is expected relative to the incorporation in plants lacking the LPAAT encoding transgenes. Oil content is also improved in the seeds, particularly for seeds producing higher levels of DHA, counteracting the trend seen in Arabidopsis seed as described in Example 2.

[0748] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0749] The present application claims priority from U.S. 61/660,392 filed 15 Jun. 2012, U.S. 61/663,344 filed 22 Jun. 2012, U.S. 61/697,676 filed 6 Sep. 2012 and U.S. 61/782,680 filed 14 Mar. 2013, the entire contents of each of which are incorporated herein by reference.

[0750] All publications discussed and/or referenced herein are incorporated herein in their entirety.

[0751] This application incorporates herein by reference U.S. 61/660,392 filed 15 Jun. 2012, U.S. 61/663,344 filed 22 Jun. 2012 and U.S. 61/697,676 filed 6 Sep. 2012.

[0752] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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Sequence CWU 1

1

72121527DNAArtificial SequencepJP3416-GA7 nucleotide sequence. 1tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180tcacgacgtt gtaaaacggg cgccccgcgg aaagcttgcg gccgcccgat ctagtaacat 240agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc 300gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat 360gcattacatg ttaattatta cgtgcttaac gtaattcaac agaaattata tgataatcat 420cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc 480ggcgcgcctc attagtgagc cttctcagcc tttccgttaa cgtagtagtg ctgtcccacc 540ttatcaaggt tagagaaagt agccttccaa gcaccgtagt aagagagcac cttgtagttg 600agtccccact tcttagcgaa aggaacgaat cttctgctaa cctcaggctg tctgaattga 660ggcatatcag ggaagaggtg gtggataacc tgacagttaa ggtatcccat aagccagttc 720acgtatcctc tagaaggatc gatatcaacg gtgtgatcaa cagcgtagtt aacccaagaa 780aggtgcttat cagatggaac aacagggagg tgagtatgag aagtagagaa gtgagcgaaa 840aggtacatgt aagcgatcca gtttccgaaa gtgaaccacc agtaagcaac aggccaagag 900tatccagtag caagcttgat aacagcggtt ctaacaacat gagaaacgag catccaagaa 960gcctcttcgt agttcttctt acggagaact tgtctagggt ggagaacgta gatccagaaa 1020gcttgaacaa gaagtccaga ggtaacagga acgaaagtcc aagcttgaag tctagcccaa 1080gctctagaga atcctctagg tctgttatcc tcaacagcag tgttgaagaa agccacagca 1140ggagtggtat caagatccat atcgtgtcta accttttgag gggtagcatg gtgcttgtta 1200tgcatctggt tccacatctc accagaagta gaaagtccga atccacaagt catagcctga 1260agtctcttgt ccacgtaaac agatccggta agagagttat gtccaccctc atgttgaacc 1320catccacatc tagctccgaa gaaagcaccg taaacaacag aagcaatgat agggtatcca 1380gcgtacataa gagcagttcc aagagcgaat gtagcaagaa gctcgagaag tctgtaagcc 1440acatgggtga tagaaggctt gaagaatcca tctctctcaa gctcagcacg ccatctagcg 1500aaatcctcaa gcataggagc atcctcagac tcagatctct tgatctcagc aggtctagaa 1560ggcaaagctc taagcatctt ccaagccttg agagaacgca tgtggaattc tttgaaagcc 1620tcagtagcat cagcaccagt gttagcaagc atgtagaaga tcacagatcc accagggtgc 1680ttgaagttag tcacatcgta ctcaacgtcc tcaactctaa cccatctagt ctcgaaagta 1740gcagcaagct catgaggctc aagagtctta agatcaacag gagcagtaga agcatcctta 1800gcatcaagag cctcagcaga agatttagac ctggtaagtg gagatctagg agaagatctt 1860ccatcagtct taggagggca catggtatgg taattgtaaa tgtaattgta atgttgtttg 1920ttgtttgttg ttgttggtaa ttgttgtaaa agatcctcgt gtatgttttt aatcttgttt 1980gtatcgatga gttttggttt gagtaaagag tgaagcggat gagttaattt ataggctata 2040aaggagattt gcatggcgat cacgtgtaat aatgcatgca cgcatgtgat tgtatgtgtg 2100tgctgtgaga gagaagctct taggtgtttg aagggagtga caagtggcga agaaaaacaa 2160ttctccgcgg ctgcatgcta tgtgtaacgt gtagctaatg ttctggcatg gcatcttatg 2220aacgattctt tttaaaaaca aggtaaaaac ttaacttcat aaaattaaaa aaaaaaacgt 2280ttactaagtt ggtttaaaag gggatgagac tagtagattg gttggttggt ttccatgtac 2340cagaaggctt accctattag ttgaaagttg aaactttgtt ccctactcaa ttcctagttg 2400tgtaaatgta tgtatatgta atgtgtataa aacgtagtac ttaaatgact aggagtggtt 2460cttgagaccg atgagagatg ggagcagaac taaagatgat gacataatta agaacgaatt 2520tgaaaggctc ttaggtttga atcctattcg agaatgtttt tgtcaaagat agtggcgatt 2580ttgaaccaaa gaaaacattt aaaaaatcag tatccggtta cgttcatgca aatagaaagt 2640ggtctaggat ctgattgtaa ttttagactt aaagagtctc ttaagattca atcctggctg 2700tgtacaaaac tacaaataat atattttaga ctatttggcc ttaactaaac ttccactcat 2760tatttactga ggttagagaa tagacttgcg aataaacaca ttcccgagaa atactcatga 2820tcccataatt agtcagaggg tatgccaatc agatctaaga acacacattc cctcaaattt 2880taatgcacat gtaatcatag tttagcacaa ttcaaaaata atgtagtatt aaagacagaa 2940atttgtagac ttttttttgg cgttaaaaga agactaagtt tatacgtaca ttttatttta 3000agtggaaaac cgaaattttc catcgaaata tatgaattta gtatatatat ttctgcaatg 3060tactattttg ctattttggc aactttcagt ggactactac tttattacaa tgtgtatgga 3120tgcatgagtt tgagtataca catgtctaaa tgcatgcttt gtaaaacgta acggaccaca 3180aaagaggatc catacaaata catctcatag cttcctccat tattttccga cacaaacaga 3240gcattttaca acaattacca acaacaacaa acaacaaaca acattacaat tacatttaca 3300attaccatac catggaattc gcccagcctc ttgttgctat ggctcaagag caatacgctg 3360ctatcgatgc tgttgttgct cctgctatct tctctgctac tgattctatc ggatggggac 3420ttaagcctat ctcttctgct actaaggact tgcctcttgt tgagtctcct acacctctca 3480tcctttcttt gcttgcttac ttcgctatcg ttggatctgg actcgtttac agaaaggttt 3540tccctagaac cgtgaaggga caagatccat tccttttgaa ggctcttatg cttgctcaca 3600acgtgttcct tatcggactt tctctttaca tgtgcctcaa gcttgtgtac gaggcttacg 3660ttaacaagta ctctttctgg ggaaacgctt acaaccctgc tcaaactgag atggctaagg 3720ttatctggat cttctacgtg agcaagatct acgagttcat ggataccttc atcatgctcc 3780tcaagggaaa tgttaaccag gttagcttcc ttcacgttta ccatcacgga tctatctctg 3840gaatctggtg gatgattact tacgctgctc ctggtggtga tgcttacttc tctgctgctc 3900ttaactcttg ggttcacgtg tgtatgtaca cctactattt tatggctgcc gtgcttccta 3960aggacgagaa aactaagaga aagtacctct ggtggggaag ataccttact caaatgcaga 4020tgttccagtt cttcatgaac cttctccagg ctgtttacct tctctactct tcatctcctt 4080accctaagtt tatcgctcag ctcctcgtgg tgtacatggt tactcttctc atgcttttcg 4140gaaacttcta ctacatgaag caccacgcta gcaagtgatg aggcgcgccg ggccgccgcc 4200atgtgacaga tcgaaggaag aaagtgtaat aagacgactc tcactactcg atcgctagtg 4260attgtcattg ttatatataa taatgttatc tttcacaact tatcgtaatg catgtgaaac 4320tataacacat taatcctact tgtcatatga taacactctc cccatttaaa actcttgtca 4380atttaaagat ataagattct ttaaatgatt aaaaaaaata tattataaat tcaatcactc 4440ctactaataa attattaatt attatttatt gattaaaaaa atacttatac taatttagtc 4500tgaatagaat aattagattc tagtctcatc cccttttaaa ccaacttagt aaacgttttt 4560ttttttaatt ttatgaagtt aagtttttac cttgttttta aaaagaatcg ttcataagat 4620gccatgccag aacattagct acacgttaca catagcatgc agccgcggag aattgttttt 4680cttcgccact tgtcactccc ttcaaacacc taagagcttc tctctcacag cacacacata 4740caatcacatg cgtgcatgca ttattacacg tgatcgccat gcaaatctcc tttatagcct 4800ataaattaac tcatccgctt cactctttac tcaaaccaaa actcatcgat acaaacaaga 4860ttaaaaacat acacgaggat cttttacaac aattaccaac aacaacaaac aacaaacaac 4920attacaatta catttacaat taccatacca tgcctccaag ggactcttac tcttatgctg 4980ctcctccttc tgctcaactt cacgaagttg atactcctca agagcacgac aagaaagagc 5040ttgttatcgg agatagggct tacgatgtta ccaacttcgt taagagacac cctggtggaa 5100agatcattgc ttaccaagtt ggaactgatg ctaccgatgc ttacaagcag ttccatgtta 5160gatctgctaa ggctgacaag atgcttaagt ctcttccttc tcgtcctgtt cacaagggat 5220actctccaag aagggctgat cttatcgctg atttccaaga gttcaccaag caacttgagg 5280ctgagggaat gttcgagcct tctcttcctc atgttgctta cagacttgct gaggttatcg 5340ctatgcatgt tgctggtgct gctcttatct ggcatggata cactttcgct ggaatcgcta 5400tgcttggagt tgttcaggga agatgtggat ggcttatgca tgagggtgga cattactctc 5460tcactggaaa cattgctttc gacagagcta tccaagttgc ttgttacgga cttggatgtg 5520gaatgtctgg tgcttggtgg cgtaaccagc ataacaagca ccatgctact cctcaaaagc 5580ttcagcacga tgttgatctt gatacccttc ctctcgttgc tttccatgag agaatcgctg 5640ctaaggttaa gtctcctgct atgaaggctt ggctttctat gcaagctaag cttttcgctc 5700ctgttaccac tcttcttgtt gctcttggat ggcagcttta ccttcatcct agacacatgc 5760tcaggactaa gcactacgat gagcttgcta tgctcggaat cagatacgga cttgttggat 5820accttgctgc taactacggt gctggatacg ttctcgcttg ttaccttctt tacgttcagc 5880ttggagctat gtacatcttc tgcaacttcg ctgtttctca tactcacctc cctgttgttg 5940agcctaacga gcatgctact tgggttgagt acgctgctaa ccacactact aactgttctc 6000catcttggtg gtgtgattgg tggatgtctt accttaacta ccagatcgag caccaccttt 6060acccttctat gcctcaattc agacacccta agatcgctcc tagagttaag cagcttttcg 6120agaagcacgg acttcactac gatgttagag gatacttcga ggctatggct gatactttcg 6180ctaaccttga taacgttgcc catgctcctg agaagaaaat gcagtaatga gatcgttcaa 6240acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca 6300tataatttct gttgaattac gttaagcacg taataattaa catgtaatgc atgacgttat 6360ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa 6420acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag 6480atcggtcgat taaaaatccc aattatattt ggtctaattt agtttggtat tgagtaaaac 6540aaattcgaac caaaccaaaa tataaatata tagtttttat atatatgcct ttaagacttt 6600ttatagaatt ttctttaaaa aatatctaga aatatttgcg actcttctgg catgtaatat 6660ttcgttaaat atgaagtgct ccatttttat taactttaaa taattggttg tacgatcact 6720ttcttatcaa gtgttactaa aatgcgtcaa tctctttgtt cttccatatt catatgtcaa 6780aatctatcaa aattcttata tatctttttc gaatttgaag tgaaatttcg ataatttaaa 6840attaaataga acatatcatt atttaggtat catattgatt tttatactta attactaaat 6900ttggttaact ttgaaagtgt acatcaacga aaaattagtc aaacgactaa aataaataaa 6960tatcatgtgt tattaagaaa attctcctat aagaatattt taatagatca tatgtttgta 7020aaaaaaatta atttttacta acacatatat ttacttatca aaaatttgac aaagtaagat 7080taaaataata ttcatctaac aaaaaaaaaa ccagaaaatg ctgaaaaccc ggcaaaaccg 7140aaccaatcca aaccgatata gttggtttgg tttgattttg atataaaccg aaccaactcg 7200gtccatttgc acccctaatc ataatagctt taatatttca agatattatt aagttaacgt 7260tgtcaatatc ctggaaattt tgcaaaatga atcaagccta tatggctgta atatgaattt 7320aaaagcagct cgatgtggtg gtaatatgta atttacttga ttctaaaaaa atatcccaag 7380tattaataat ttctgctagg aagaaggtta gctacgattt acagcaaagc cagaatacaa 7440agaaccataa agtgattgaa gctcgaaata tacgaaggaa caaatatttt taaaaaaata 7500cgcaatgact tggaacaaaa gaaagtgata tattttttgt tcttaaacaa gcatcccctc 7560taaagaatgg cagttttcct ttgcatgtaa ctattatgct cccttcgtta caaaaatttt 7620ggactactat tgggaacttc ttctgaaaat agtgatagaa cccacacgag catgtgcttt 7680ccatttaatt ttaaaaacca agaaacatac atacataaca ttccatcagc ctctctctct 7740ttttattacg gttaatgact taaaacacat cttattatcc catccttaac acctagcagt 7800gtctttatac gatctcatcg atcaccactt caaaaccatg cagactgctg ctgcccctgg 7860agctggcatc ggctaggctg ggtgccgcac tgtcccggaa ggtccctagc gacttgttta 7920gattgatggg accacctctc aacttcctgc tgctgtccct gctgctggat gtcctgcctc 7980atctggccga ttgcacgctc cagtcccctg catgtgcact cgctcctcaa ttgcttaaga 8040tcatcgcagc agctatcgaa gtgctggctc tgttgccctc ctccacggcc ttggttgtag 8100tagtagctgc cgccgccctt ctggactttt tcccacagga accgccgaat aattcgatag 8160aaccacacga gcatgtgctt tcatttattt taaaaaccaa gaaacataca taacatttca 8220tcagcctctc tctctctctc tctctctctc tctctctctc tctctctctc tctctcttta 8280ttacagctgt tacactaact taaaacacat tcatctcatt attattatta ttatccatcc 8340ttaacaccta gcagtgtctt tgtacgatct cataatcgat caccccttca tcaggtatcc 8400ttaggcttca ctccaacgtt gttgcagtta cggaacatgt acacaccatc atggttctca 8460acgaactggc aagatctcca agttttccaa aggctaaccc acatgttctc atcggtgtgt 8520ctgtagtgct ctcccataac tttcttgatg cactcggtag cttctctagc atggtagaat 8580gggatccttg aaacgtagtg atggagcaca tgagtctcga tgatgtcatg gaagatgatt 8640ccgaggattc cgaactctct atcgatagta gcagcagcac ccttagcgaa agtccactct 8700tgagcatcgt aatgaggcat agaagaatcg gtgtgctgaa ggaaggtaac gaaaacaagc 8760cagtggttaa caaggatcca aggacagaac catgtgatga aagtaggcca gaatccgaaa 8820accttgtaag cggtgtaaac agaagtgagg gtagcaagga ttccaagatc agaaagaacg 8880atgtaccagt agtccttctt atcgaaaaca gggctagaag gccagtagtg agacttgaag 8940aacttagaaa caccagggta aggttgtcca gtagcgttag tagcaaggta aagagaaagt 9000cctccaagct gttggaacaa gagagcgaaa acagagtaga taggagtttc ctcagcgata 9060tcgtgaaggc tggtaacttg gtgcttctct ttgaattcct cggcggtgta aggaacgaaa 9120accatatctc tggtcatgtg tccagtagcc ttatggtgct tagcatgaga gaacttccag 9180ctgaagtaag gaaccataac aagagagtgg agaacccatc caacggtatc gttaacccat 9240ccgtagttag agaaagcaga atgtccacac tcatgtccaa ggatccagat tccgaatccg 9300aaacaagaga tagagaacac gtaagcagac caagcagcga atctaaggaa ttcgttaggg 9360agaagaggga tgtaggtaag tccaacgtaa gcgatagcag agatagccac gatatctctc 9420accacgtaag acatagactt cacgagagat ctctcgtaac agtgcttagg gatagcgtca 9480aggatatcct tgatggtgta atctggcacc ttgaaaacgt ttccgaaggt atcgatagcg 9540gtcttttgct gcttgaaaga tgcaacgttt ccagaacgcc taacggtctt agtagatccc 9600tcaaggatct cagatccaga cacggtaacc ttagacatgg tatggtaatt gtaaatgtaa 9660ttgtaatgtt gtttgttgtt tgttgttgtt ggtaattgtt gtaaaatttt tggtggtgat 9720tggttcttta aggtgtgaga gtgagttgtg agttgtgtgg tgggtttggt gagattgggg 9780atggtgggtt tatatagtgg agactgagga atggggtcgt gagtgttaac tttgcatggg 9840ctacacgtgg gttcttttgg gcttacacgt agtattattc atgcaaatgc agccaataca 9900tatacggtat tttaataatg tgtgggaata caatatgccg agtattttac taattttggc 9960aatgacaagt gtacatttgg attatcttac ttggcctctc ttgctttaat ttggattatt 10020tttattctct taccttggcc gttcatattc acatccctaa aggcaagaca gaattgaatg 10080gtggccaaaa attaaaacga tggatatgac ctacatagtg taggatcaat taacgtcgaa 10140ggaaaatact gattctctca agcatacgga caagggtaaa taacatagtc accagaacat 10200aataaacaaa aagtgcagaa gcaagactaa aaaaattagc tatggacatt caggttcata 10260ttggaaacat cattatccta gtcttgtgac catccttcct cctgctctag ttgagaggcc 10320ttgggactaa cgagaggtca gttgggatag cagatcctta tcctggacta gcctttctgg 10380tgtttcagag tcttcgtgcc gccgtctaca tctatctcca ttaggtctga agatgactct 10440tcacaccaac gacgtttaag gtctctatcc tactcctagc ttgcaatacc tggcttgcaa 10500tacctggagc atcgtgcacg atgattggat actgtggagg aggagtgttt gctgatttag 10560agctcccggt tgggtgattt gacttcgatt tcagtttagg cttgttgaaa tttttcaggt 10620tccattgtga agcctttaga gcttgagctt ccttccatgt taatgccttg atcgaatact 10680cctagagaaa agggaagtcg atctctgagt attgaaatcg aagtgcacat tttttttcaa 10740cgtgtccaat caatccacaa acaaagcaga agacaggtaa tctttcatac ttatactgac 10800aagtaatagt cttaccgtca tgcataataa cgtctcgttc cttcaagagg ggttttccga 10860catccataac gacccgaagc ctcatgaaag cattagggaa gaacttttgg ttcttcttgt 10920catggccttt ataggtgtca gccgagctcg ccaattcccg tccgactggc tccgcaaaat 10980attcgaacgg caagttatgg acttgcaacc ataactccac ggtattgagc aggacctatt 11040gtgaagactc atctcatgga gcttcagaat gtggttgtca gcaaaccaat gaccgaaatc 11100catcacatga cggacgtcca gtgggtgagc gaaacgaaac aggaagcgcc tatctttcag 11160agtcgtgagc tccacaccgg attccggcaa ctacgtgttg ggcaggcttc gccgtattag 11220agatatgttg aggcagaccc atctgtgcca ctcgtacaat tacgagagtt gttttttttg 11280tgattttcct agtttctcgt tgatggtgag ctcatattct acatcgtatg gtctctcaac 11340gtcgtttcct gtcatctgat atcccgtcat ttgcatccac gtgcgccgcc tcccgtgcca 11400agtccctagg tgtcatgcac gccaaattgg tggtggtgcg ggctgccctg tgcttcttac 11460cgatgggtgg aggttgagtt tgggggtctc cgcggcgatg gtagtgggtt gacggtttgg 11520tgtgggttga cggcattgat caatttactt cttgcttcaa attctttggc agaaaacaat 11580tcattagatt agaactggaa accagagtga tgagacggat taagtcagat tccaacagag 11640ttacatctct taagaaataa tgtaacccct ttagacttta tatatttgca attaaaaaaa 11700taatttaact tttagacttt atatatagtt ttaataacta agtttaacca ctctattatt 11760tatatcgaaa ctatttgtat gtctcccctc taaataaact tggtattgtg tttacagaac 11820ctataatcaa ataatcaata ctcaactgaa gtttgtgcag ttaattgaag ggattaacgg 11880ccaaaatgca ctagtattat caaccgaata gattcacact agatggccat ttccatcaat 11940atcatcgccg ttcttcttct gtccacatat cccctctgaa acttgagaga cacctgcact 12000tcattgtcct tattacgtgt tacaaaatga aacccatgca tccatgcaaa ctgaagaatg 12060gcgcaagaac ccttcccctc catttcttat gtggcgacca tccatttcac catctcccgc 12120tataaaacac ccccatcact tcacctagaa catcatcact acttgcttat ccatccaaaa 12180gatacccact tttacaacaa ttaccaacaa caacaaacaa caaacaacat tacaattaca 12240tttacaatta ccataccatg ccacctagcg ctgctaagca aatgggagct tctactggtg 12300ttcatgctgg tgttactgac tcttctgctt tcaccagaaa ggatgttgct gatagacctg 12360atctcaccat cgttggagat tctgtttacg atgctaaggc tttcagatct gagcatcctg 12420gtggtgctca tttcgtttct ttgttcggag gaagagatgc tactgaggct ttcatggaat 12480accatagaag ggcttggcct aagtctagaa tgtctagatt ccacgttgga tctcttgctt 12540ctactgagga acctgttgct gctgatgagg gataccttca actttgtgct aggatcgcta 12600agatggtgcc ttctgtttct tctggattcg ctcctgcttc ttactgggtt aaggctggac 12660ttatccttgg atctgctatc gctcttgagg cttacatgct ttacgctgga aagagacttc 12720tcccttctat cgttcttgga tggcttttcg ctcttatcgg tcttaacatc cagcatgatg 12780ctaaccatgg tgctttgtct aagtctgctt ctgttaacct tgctcttgga ctttgtcagg 12840attggatcgg aggatctatg atcctttggc ttcaagagca tgttgttatg caccacctcc 12900acactaacga tgttgataag gatcctgatc aaaaggctca cggtgctctt agactcaagc 12960ctactgatgc ttggtcacct atgcattggc ttcagcatct ttaccttttg cctggtgaga 13020ctatgtacgc tttcaagctt ttgttcctcg acatctctga gcttgttatg tggcgttggg 13080agggtgagcc tatctctaag cttgctggat acctctttat gccttctttg cttctcaagc 13140ttaccttctg ggctagattc gttgctttgc ctctttacct tgctccttct gttcatactg 13200ctgtgtgtat cgctgctact gttatgactg gatctttcta cctcgctttc ttcttcttca 13260tctcccacaa cttcgagggt gttgcttctg ttggacctga tggatctatc acttctatga 13320ctagaggtgc tagcttcctt aagagacaag ctgagacttc ttctaacgtt ggaggacctc 13380ttcttgctac tcttaacggt ggactcaact accaaattga gcatcacttg ttccctagag 13440ttcaccatgg attctaccct agacttgctc ctcttgttaa ggctgagctt gaggctagag 13500gaatcgagta caagcactac cctactatct ggtctaacct tgcttctacc ctcagacata 13560tgtacgctct tggaagaagg cctagatcta aggctgagta atgacaagct tatgtgacgt 13620gaaataataa cggtaaaata tatgtaataa taataataat aaagccacaa agtgagaatg 13680aggggaaggg gaaatgtgta atgagccagt agccggtggt gctaattttg tatcgtattg 13740tcaataaatc atgaattttg tggtttttat gtgttttttt aaatcatgaa ttttaaattt 13800tataaaataa tctccaatcg gaagaacaac attccatatc catgcatgga tgtttcttta 13860cccaaatcta gttcttgaga ggatgaagca tcaccgaaca gttctgcaac tatccctcaa 13920aagctttaaa atgaacaaca aggaacagag caacgttcca aagatcccaa acgaaacata 13980ttatctatac taatactata ttattaatta ctactgcccg gaatcacaat ccctgaatga 14040ttcctattaa ctacaagcct tgttggcggc ggagaagtga tcggcgcggc gagaagcagc 14100ggactcggag acgaggcctt ggaagatctg agtcgaacgg gcagaatcag tattttcctt 14160cgacgttaat tgatcctaca ctatgtaggt catatccatc gttttaattt ttggccacca 14220ttcaattctg tcttgccttt agggatgtga atatgaacgg ccaaggtaag agaataaaaa 14280taatccaaat taaagcaaga gaggccaagt aagataatcc aaatgtacac ttgtcattgc 14340caaaattagt aaaatactcg gcatattgta ttcccacaca ttattaaaat accgtatatg 14400tattggctgc atttgcatga ataatactac gtgtaagccc aaaagaaccc acgtgtagcc 14460catgcaaagt taacactcac gaccccattc ctcagtctcc actatataaa cccaccatcc 14520ccaatctcac caaacccacc acacaactca caactcactc tcacacctta aagaaccaat 14580caccaccaaa aattttacaa caattaccaa caacaacaaa caacaaacaa cattacaatt 14640acatttacaa ttaccatacc atgagcgctg ttaccgttac tggatctgat cctaagaaca 14700gaggatcttc tagcaacacc gagcaagagg ttccaaaagt tgctatcgat accaacggaa 14760acgtgttctc tgttcctgat ttcaccatca aggacatcct tggagctatc cctcatgagt 14820gttacgagag aagattggct acctctctct actacgtgtt cagagatatc ttctgcatgc 14880ttaccaccgg ataccttacc cataagatcc tttaccctct cctcatctct tacacctcta 14940acagcatcat caagttcact ttctgggccc tttacactta cgttcaagga cttttcggaa 15000ccggaatctg

ggttctcgct catgagtgtg gacatcaagc tttctctgat tacggaatcg 15060tgaacgattt cgttggatgg acccttcact cttaccttat ggttccttac ttcagctgga 15120agtactctca tggaaagcac cataaggcta ctggacacat gaccagagat atggttttcg 15180ttcctgccac caaagaggaa ttcaagaagt ctaggaactt cttcggtaac ctcgctgagt 15240actctgagga ttctccactt agaacccttt acgagcttct tgttcaacaa cttggaggat 15300ggatcgctta cctcttcgtt aacgttacag gacaacctta ccctgatgtt ccttcttgga 15360aatggaacca cttctggctt acctctccac ttttcgagca aagagatgct ctctacatct 15420tcctttctga tcttggaatc ctcacccagg gaatcgttct tactctttgg tacaagaaat 15480tcggaggatg gtcccttttc atcaactggt tcgttcctta catctgggtt aaccactggc 15540tcgttttcat cacattcctt cagcacactg atcctactat gcctcattac aacgctgagg 15600aatggacttt cgctaagggt gctgctgcta ctatcgatag aaagttcgga ttcatcggac 15660ctcacatctt ccatgatatc atcgagactc atgtgcttca ccactactgt tctaggatcc 15720cattctacaa cgctagacct gcttctgagg ctatcaagaa agttatggga aagcactaca 15780ggtctagcga cgagaacatg tggaagtcac tttggaagtc tttcaggtct tgccaatacg 15840ttgacggtga taacggtgtt ctcatgttcc gtaacatcaa caactgcgga gttggagctg 15900ctgagaagta atgaaggggt gatcgattat gagatcgtac aaagacactg ctaggtgtta 15960aggatggata ataataataa taatgagatg aatgtgtttt aagttagtgt aacagctgta 16020ataaagagag agagagagag agagagagag agagagagag agagagagag agagaggctg 16080atgaaatgtt atgtatgttt cttggttttt aaaataaatg aaagcacatg ctcgtgtggt 16140tctatcgaat tattcggcgg ttcctgtggg aaaaagtcca gaagggccgc cgcagctact 16200actacaacca aggccgtgga ggagggcaac agagccagca cttcgatagc tgctgcgatg 16260atcttaagca attgaggagc gagtgcacat gcaggggact ggagcgtgca atcggccaga 16320tgaggcagga catccagcag cagggacagc agcaggaagt tgagaggtgg tcccatcaat 16380ctaaacaagt cgctagggac cttccgggac agtgcggcac ccagcctagc cgatgccagc 16440tccaggggca gcagcagtct gcatggtttt gaagtggtga tcgatgagat cgtataaaga 16500cactgctagg tgttaaggat gggataataa gatgtgtttt aagtcattaa ccgtaataaa 16560aagagagaga ggctgatgga atgttatgta tgtatgtttc ttggttttta aaattaaatg 16620gaaagcacat gctcgtgtgg gttctatctc gattaaaaat cccaattata tttggtctaa 16680tttagtttgg tattgagtaa aacaaattcg aaccaaacca aaatataaat atatagtttt 16740tatatatatg cctttaagac tttttataga attttcttta aaaaatatct agaaatattt 16800gcgactcttc tggcatgtaa tatttcgtta aatatgaagt gctccatttt tattaacttt 16860aaataattgg ttgtacgatc actttcttat caagtgttac taaaatgcgt caatctcttt 16920gttcttccat attcatatgt caaaatctat caaaattctt atatatcttt ttcgaatttg 16980aagtgaaatt tcgataattt aaaattaaat agaacatatc attatttagg tatcatattg 17040atttttatac ttaattacta aatttggtta actttgaaag tgtacatcaa cgaaaaatta 17100gtcaaacgac taaaataaat aaatatcatg tgttattaag aaaattctcc tataagaata 17160ttttaataga tcatatgttt gtaaaaaaaa ttaattttta ctaacacata tatttactta 17220tcaaaaattt gacaaagtaa gattaaaata atattcatct aacaaaaaaa aaaccagaaa 17280atgctgaaaa cccggcaaaa ccgaaccaat ccaaaccgat atagttggtt tggtttgatt 17340ttgatataaa ccgaaccaac tcggtccatt tgcaccccta atcataatag ctttaatatt 17400tcaagatatt attaagttaa cgttgtcaat atcctggaaa ttttgcaaaa tgaatcaagc 17460ctatatggct gtaatatgaa tttaaaagca gctcgatgtg gtggtaatat gtaatttact 17520tgattctaaa aaaatatccc aagtattaat aatttctgct aggaagaagg ttagctacga 17580tttacagcaa agccagaata caaagaacca taaagtgatt gaagctcgaa atatacgaag 17640gaacaaatat ttttaaaaaa atacgcaatg acttggaaca aaagaaagtg atatattttt 17700tgttcttaaa caagcatccc ctctaaagaa tggcagtttt cctttgcatg taactattat 17760gctcccttcg ttacaaaaat tttggactac tattgggaac ttcttctgaa aatagtcctg 17820caggctagta gattggttgg ttggtttcca tgtaccagaa ggcttaccct attagttgaa 17880agttgaaact ttgttcccta ctcaattcct agttgtgtaa atgtatgtat atgtaatgtg 17940tataaaacgt agtacttaaa tgactaggag tggttcttga gaccgatgag agatgggagc 18000agaactaaag atgatgacat aattaagaac gaatttgaaa ggctcttagg tttgaatcct 18060attcgagaat gtttttgtca aagatagtgg cgattttgaa ccaaagaaaa catttaaaaa 18120atcagtatcc ggttacgttc atgcaaatag aaagtggtct aggatctgat tgtaatttta 18180gacttaaaga gtctcttaag attcaatcct ggctgtgtac aaaactacaa ataatatatt 18240ttagactatt tggccttaac taaacttcca ctcattattt actgaggtta gagaatagac 18300ttgcgaataa acacattccc gagaaatact catgatccca taattagtca gagggtatgc 18360caatcagatc taagaacaca cattccctca aattttaatg cacatgtaat catagtttag 18420cacaattcaa aaataatgta gtattaaaga cagaaatttg tagacttttt tttggcgtta 18480aaagaagact aagtttatac gtacatttta ttttaagtgg aaaaccgaaa ttttccatcg 18540aaatatatga atttagtata tatatttctg caatgtacta ttttgctatt ttggcaactt 18600tcagtggact actactttat tacaatgtgt atggatgcat gagtttgagt atacacatgt 18660ctaaatgcat gctttgtaaa acgtaacgga ccacaaaaga ggatccatac aaatacatct 18720catagcttcc tccattattt tccgacacaa acagagcatt ttacaacaat taccaacaac 18780aacaaacaac aaacaacatt acaattacat ttacaattac cataccatgg cctctatcgc 18840tatccctgct gctcttgctg gaactcttgg atacgttacc tacaatgtgg ctaaccctga 18900tatcccagct tctgagaaag ttcctgctta cttcatgcag gttgagtact ggggacctac 18960tatcggaact attggatacc tcctcttcat ctacttcgga aagcgtatca tgcagaacag 19020atctcaacct ttcggactca agaacgctat gctcgtttac aacttctacc agaccttctt 19080caacagctac tgcatctacc ttttcgttac ttctcatagg gctcagggac ttaaggtttg 19140gggaaacatc cctgatatga ctgctaactc ttggggaatc tctcaggtta tctggcttca 19200ctacaacaac aagtacgttg agcttctcga caccttcttc atggtgatga ggaagaagtt 19260cgaccagctt tctttccttc acatctacca ccacactctt ctcatctggt catggttcgt 19320tgttatgaag cttgagcctg ttggagattg ctacttcgga tcttctgtta acaccttcgt 19380gcacgtgatc atgtactctt actacggact tgctgctctt ggagttaact gtttctggaa 19440gaagtacatc acccagatcc agatgcttca gttctgtatc tgtgcttctc actctatcta 19500caccgcttac gttcagaata ccgctttctg gcttccttac cttcaactct gggttatggt 19560gaacatgttc gttctcttcg ccaacttcta ccgtaagagg tacaagtcta agggtgctaa 19620gaagcagtga taagggccgc cgccatgtga cagatcgaag gaagaaagtg taataagacg 19680actctcacta ctcgatcgct agtgattgtc attgttatat ataataatgt tatctttcac 19740aacttatcgt aatgcatgtg aaactataac acattaatcc tacttgtcat atgataacac 19800tctccccatt taaaactctt gtcaatttaa agatataaga ttctttaaat gattaaaaaa 19860aatatattat aaattcaatc actcctacta ataaattatt aattattatt tattgattaa 19920aaaaatactt atactaattt agtctgaata gaataattag attctagcct gcagggcggc 19980cgcggatccc atggagtcaa agattcaaat agaggaccta acagaactcg ccgtaaagac 20040tggcgaacag ttcatacaga gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa 20100catggtggag cacgacacac ttgtctactc caaaaatatc aaagatacag tctcagaaga 20160ccaaagggca attgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca 20220ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa 20280atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg acagtggtcc 20340caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc 20400ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca 20460ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg agagaacacg 20520ggggactgaa ttaaatatga gccctgagag gcgtcctgtt gaaatcagac ctgctactgc 20580tgctgatatg gctgctgttt gtgatatcgt gaaccactac atcgagactt ctaccgttaa 20640cttcagaact gagcctcaaa ctcctcaaga gtggatcgat gatcttgaga gactccaaga 20700tagataccct tggcttgttg ctgaggttga gggtgttgtt gctggaatcg cttacgctgg 20760accttggaag gctagaaacg cttacgattg gactgttgag tctaccgttt acgtttcaca 20820cagacatcag agacttggac ttggatctac cctttacact caccttctca agtctatgga 20880agctcaggga ttcaagtctg ttgttgctgt tatcggactc cctaacgatc cttctgttag 20940acttcatgag gctcttggat acactgctag aggaactctt agagctgctg gatacaagca 21000cggtggatgg catgatgttg gattctggca aagagatttc gagcttcctg ctcctcctag 21060acctgttaga ccagttactc agatctgaat ttgcgtgatc gttcaaacat ttggcaataa 21120agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 21180aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 21240tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 21300gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatca ctagtgatgt 21360acggttaaaa ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag 21420cgtcaatttg tttacaccac aatatatcct gccaccagcc agccaacagc tccccgaccg 21480gcagctcggc acaaaatcac cactcgatac aggcagccca tcagtcc 21527223512DNAArtificial SequencepGA7- mod_B nucleotide sequence 2tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180tcacgacgtt gtaaaacggg cgccccgcgg aaagcttgcg gccgcggtac cgcccgttcg 240actcagatct tccaaggcct cgtctccgag tccgctgctt ctcgccgcgc cgatcacttc 300tccgccgcca acaaggcttg tagttaatag gaatcattca gggattgtga ttccgggcag 360tagtaattaa taatatagta ttagtataga taatatgttt cgtttgggat ctttggaacg 420ttgctctgtt ccttgttgtt cattttaaag cttttgaggg atagttgcag aactgttcgg 480tgatgcttca tcctctcaag aactagattt gggtaaagaa acatccatgc atggatatgg 540aatgttgttc ttccgattgg agattatttt ataaaattta aaattcatga tttaaaaaaa 600cacataaaaa ccacaaaatt catgatttat tgacaatacg atacaaaatt agcaccaccg 660gctactggct cattacacat ttccccttcc cctcattctc actttgtggc tttattatta 720ttattattac atatatttta ccgttattat ttcacgtcac ataagcttgt taattaatca 780ttagtgagcc ttctcagcct ttccgttaac gtagtagtgc tgtcccacct tatcaaggtt 840agagaaagta gccttccaag caccgtagta agagagcacc ttgtagttga gtccccactt 900cttagcgaaa ggaacgaatc ttctgctaac ctcaggctgt ctgaattgag gcatatcagg 960gaagaggtgg tggataacct gacagttaag gtatcccata agccagttca cgtatcctct 1020agaaggatcg atatcaacgg tgtgatcaac agcgtagtta acccaagaaa ggtgcttatc 1080agatggaaca acagggaggt gagtatgaga agtagagaag tgagcgaaaa ggtacatgta 1140agcgatccag tttccgaaag tgaaccacca gtaagcaaca ggccaagagt atccagtagc 1200aagcttgata acagcggttc taacaacatg agaaacgagc atccaagaag cctcttcgta 1260gttcttctta cggagaactt gtctagggtg gagaacgtag atccagaaag cttgaacaag 1320aagtccagag gtaacaggaa cgaaagtcca agcttgaagt ctagcccaag ctctagagaa 1380tcctctaggt ctgttatcct caacagcagt gttgaagaaa gccacagcag gagtggtatc 1440aagatccata tcgtgtctaa ccttttgagg ggtagcatgg tgcttgttat gcatctggtt 1500ccacatctca ccagaagtag aaagtccgaa tccacaagtc atagcctgaa gtctcttgtc 1560cacgtaaaca gatccggtaa gagagttatg tccaccctca tgttgaaccc atccacatct 1620agctccgaag aaagcaccgt aaacaacaga agcaatgata gggtatccag cgtacataag 1680agcagttcca agagcgaatg tagcaagaag ctcgagaagt ctgtaagcca catgggtgat 1740agaaggcttg aagaatccat ctctctcaag ctcagcacgc catctagcga aatcctcaag 1800cataggagca tcctcagact cagatctctt gatctcagca ggtctagaag gcaaagctct 1860aagcatcttc caagccttga gagaacgcat gtggaattct ttgaaagcct cagtagcatc 1920agcaccagtg ttagcaagca tgtagaagat cacagatcca ccagggtgct tgaagttagt 1980cacatcgtac tcaacgtcct caactctaac ccatctagtc tcgaaagtag cagcaagctc 2040atgaggctca agagtcttaa gatcaacagg agcagtagaa gcatccttag catcaagagc 2100ctcagcagaa gatttagacc tggtaagtgg agatctagga gaagatcttc catcagtctt 2160aggagggcac atggtatggt aattgtaaat gtaattgtaa tgttgtttgt tgtttgttgt 2220tgttggtaat tgttgtaaaa ttaattaagt gggtatcttt tggatggata agcaagtagt 2280gatgatgttc taggtgaagt gatgggggtg ttttatagcg ggagatggtg aaatggatgg 2340tcgccacata agaaatggag gggaagggtt cttgcgccat tcttcagttt gcatggatgc 2400atgggtttca ttttgtaaca cgtaataagg acaatgaagt gcaggtgtct ctcaagtttc 2460agaggggata tgtggacaga agaagaacgg cgatgatatt gatggaaatg gccatctagt 2520gtgaatctat tcggttgata atactagtgc attttggccg ttaatccctt caattaactg 2580cacaaacttc agttgagtat tgattatttg attataggtt ctgtaaacac aataccaagt 2640ttatttagag gggagacata caaatagttt cgatataaat aatagagtgg ttaaacttag 2700ttattaaaac tatatataaa gtctaaaagt taaattattt ttttaattgc aaatatataa 2760agtctaaagg ggttacatta tttcttaaga gatgtaactc tgttggaatc tgacttaatc 2820cgtctcatca ctctggtttc cagttctaat ctaatgaatt gttttctgcc aaagaatttg 2880aagcaagaag taaattgatc aatgccgtca acccacacca aaccgtcaac ccactaccat 2940cgccgcggag acccccaaac tcaacctcca cccatcggta agaagcacag ggcagcccgc 3000accaccacca atttggcgtg catgacacct agggacttgg cacgggaggc ggcgcacgtg 3060gatgcaaatg acgggatatc agatgacagg aaacgacgtt gagagaccat acgatgtaga 3120atatgagctc accatcaacg agaaactagg aaaatcacaa aaaaaacaac tctcgtaatt 3180gtacgagtgg cacagatggg tctgcctcaa catatctcta atacggcgaa gcctgcccaa 3240cacgtagttg ccggaatccg gtgtggagct cacgactctg aaagataggc gcttcctgtt 3300tcgtttcgct cacccactgg acgtccgtca tgtgatggat ttcggtcatt ggtttgctga 3360caaccacatt ctgaagctcc atgagatgag tcttcacaat aggtcctgct caataccgtg 3420gagttatggt tgcaagtcca taacttgccg ttcgaatatt ttgcggagcc agtcggacgg 3480gaattggcga gctcggctga cacctataaa ggccatgaca agaagaacca aaagttcttc 3540cctaatgctt tcatgaggct tcgggtcgtt atggatgtcg gaaaacccct cttgaaggaa 3600cgagacgtta ttatgcatga cggtaagact attacttgtc agtataagta tgaaagatta 3660cctgtcttct gctttgtttg tggattgatt ggacacgttg aaaaaaaatg tgcacttcga 3720tttcaatact cagagatcga cttccctttt ctctaggagt attcgatcaa ggcattaaca 3780tggaaggaag ctcaagctct aaaggcttca caatggaacc tgaaaaattt caacaagcct 3840aaactgaaat cgaagtcaaa tcacccaacc gggagctcta aatcagcaaa cactcctcct 3900ccacagtatc caatcatcgt gcacgatgct ccaggtattg caagccaggt attgcaagct 3960aggagtagga tagagacctt aaacgtcgtt ggtgtgaaga gtcatcttca gacctaatgg 4020agatagatgt agacggcggc acgaagactc tgaaacacca gaaaggctag tccaggataa 4080ggatctgcta tcccaactga cctctcgtta gtcccaaggc ctctcaacta gagcaggagg 4140aaggatggtc acaagactag gataatgatg tttccaatat gaacctgaat gtccatagct 4200aattttttta gtcttgcttc tgcacttttt gtttattatg ttctggtgac tatgttattt 4260acccttgtcc gtatgcttga gggtacccta gtagattggt tggttggttt ccatgtacca 4320gaaggcttac cctattagtt gaaagttgaa actttgttcc ctactcaatt cctagttgtg 4380taaatgtatg tatatgtaat gtgtataaaa cgtagtactt aaatgactag gagtggttct 4440tgagaccgat gagagatggg agcagaacta aagatgatga cataattaag aacgaatttg 4500aaaggctctt aggtttgaat cctattcgag aatgtttttg tcaaagatag tggcgatttt 4560gaaccaaaga aaacatttaa aaaatcagta tccggttacg ttcatgcaaa tagaaagtgg 4620tctaggatct gattgtaatt ttagacttaa agagtctctt aagattcaat cctggctgtg 4680tacaaaacta caaataatat attttagact atttggcctt aactaaactt ccactcatta 4740tttactgagg ttagagaata gacttgcgaa taaacacatt cccgagaaat actcatgatc 4800ccataattag tcagagggta tgccaatcag atctaagaac acacattccc tcaaatttta 4860atgcacatgt aatcatagtt tagcacaatt caaaaataat gtagtattaa agacagaaat 4920ttgtagactt ttttttggcg ttaaaagaag actaagttta tacgtacatt ttattttaag 4980tggaaaaccg aaattttcca tcgaaatata tgaatttagt atatatattt ctgcaatgta 5040ctattttgct attttggcaa ctttcagtgg actactactt tattacaatg tgtatggatg 5100catgagtttg agtatacaca tgtctaaatg catgctttgt aaaacgtaac ggaccacaaa 5160agaggatcca tacaaataca tctcatagct tcctccatta ttttccgaca caaacagagc 5220attttacaac aattaccaac aacaacaaac aacaaacaac attacaatta catttacaat 5280taccatacca tggcctctat cgctatccct gctgctcttg ctggaactct tggatacgtt 5340acctacaatg tggctaaccc tgatatccca gcttctgaga aagttcctgc ttacttcatg 5400caggttgagt actggggacc tactatcgga actattggat acctcctctt catctacttc 5460ggaaagcgta tcatgcagaa cagatctcaa cctttcggac tcaagaacgc tatgctcgtt 5520tacaacttct accagacctt cttcaacagc tactgcatct accttttcgt tacttctcat 5580agggctcagg gacttaaggt ttggggaaac atccctgata tgactgctaa ctcttgggga 5640atctctcagg ttatctggct tcactacaac aacaagtacg ttgagcttct cgacaccttc 5700ttcatggtga tgaggaagaa gttcgaccag ctttctttcc ttcacatcta ccaccacact 5760cttctcatct ggtcatggtt cgttgttatg aagcttgagc ctgttggaga ttgctacttc 5820ggatcttctg ttaacacctt cgtgcacgtg atcatgtact cttactacgg acttgctgct 5880cttggagtta actgtttctg gaagaagtac atcacccaga tccagatgct tcagttctgt 5940atctgtgctt ctcactctat ctacaccgct tacgttcaga ataccgcttt ctggcttcct 6000taccttcaac tctgggttat ggtgaacatg ttcgttctct tcgccaactt ctaccgtaag 6060aggtacaagt ctaagggtgc taagaagcag tgataaggcg cgcggcgcgc cgggccgccg 6120ccatgtgaca gatcgaagga agaaagtgta ataagacgac tctcactact cgatcgctag 6180tgattgtcat tgttatatat aataatgtta tctttcacaa cttatcgtaa tgcatgtgaa 6240actataacac attaatccta cttgtcatat gataacactc tccccattta aaactcttgt 6300caatttaaag atataagatt ctttaaatga ttaaaaaaaa tatattataa attcaatcac 6360tcctactaat aaattattaa ttattattta ttgattaaaa aaatacttat actaatttag 6420tctgaataga ataattagat tctagtctca tcccctttta aaccaactta gtaaacgttt 6480ttttttttaa ttttatgaag ttaagttttt accttgtttt taaaaagaat cgttcataag 6540atgccatgcc agaacattag ctacacgtta cacatagcat gcagccgcgg agaattgttt 6600ttcttcgcca cttgtcactc ccttcaaaca cctaagagct tctctctcac agcacacaca 6660tacaatcaca tgcgtgcatg cattattaca cgtgatcgcc atgcaaatct cctttatagc 6720ctataaatta actcatccgc ttcactcttt actcaaacca aaactcatcg atacaaacaa 6780gattaaaaac atacacgagg atcttttaca acaattacca acaacaacaa acaacaaaca 6840acattacaat tacatttaca attaccatac catgcctcca agggactctt actcttatgc 6900tgctcctcct tctgctcaac ttcacgaagt tgatactcct caagagcacg acaagaaaga 6960gcttgttatc ggagataggg cttacgatgt taccaacttc gttaagagac accctggtgg 7020aaagatcatt gcttaccaag ttggaactga tgctaccgat gcttacaagc agttccatgt 7080tagatctgct aaggctgaca agatgcttaa gtctcttcct tctcgtcctg ttcacaaggg 7140atactctcca agaagggctg atcttatcgc tgatttccaa gagttcacca agcaacttga 7200ggctgaggga atgttcgagc cttctcttcc tcatgttgct tacagacttg ctgaggttat 7260cgctatgcat gttgctggtg ctgctcttat ctggcatgga tacactttcg ctggaatcgc 7320tatgcttgga gttgttcagg gaagatgtgg atggcttatg catgagggtg gacattactc 7380tctcactgga aacattgctt tcgacagagc tatccaagtt gcttgttacg gacttggatg 7440tggaatgtct ggtgcttggt ggcgtaacca gcataacaag caccatgcta ctcctcaaaa 7500gcttcagcac gatgttgatc ttgataccct tcctctcgtt gctttccatg agagaatcgc 7560tgctaaggtt aagtctcctg ctatgaaggc ttggctttct atgcaagcta agcttttcgc 7620tcctgttacc actcttcttg ttgctcttgg atggcagctt taccttcatc ctagacacat 7680gctcaggact aagcactacg atgagcttgc tatgctcgga atcagatacg gacttgttgg 7740ataccttgct gctaactacg gtgctggata cgttctcgct tgttaccttc tttacgttca 7800gcttggagct atgtacatct tctgcaactt cgctgtttct catactcacc tccctgttgt 7860tgagcctaac gagcatgcta cttgggttga gtacgctgct aaccacacta ctaactgttc 7920tccatcttgg tggtgtgatt ggtggatgtc ttaccttaac taccagatcg agcaccacct 7980ttacccttct atgcctcaat tcagacaccc taagatcgct cctagagtta agcagctttt 8040cgagaagcac ggacttcact acgatgttag aggatacttc gaggctatgg ctgatacttt 8100cgctaacctt gataacgttg cccatgctcc tgagaagaaa atgcagtaat gagatcgttc 8160aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat 8220catataattt ctgttgaatt acgttaagca cgtaataatt aacatgtaat gcatgacgtt 8280atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga 8340aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact 8400agatcggtcg attaaaaatc ccaattatat ttggtctaat ttagtttggt attgagtaaa

8460acaaattcga accaaaccaa aatataaata tatagttttt atatatatgc ctttaagact 8520ttttatagaa ttttctttaa aaaatatcta gaaatatttg cgactcttct ggcatgtaat 8580atttcgttaa atatgaagtg ctccattttt attaacttta aataattggt tgtacgatca 8640ctttcttatc aagtgttact aaaatgcgtc aatctctttg ttcttccata ttcatatgtc 8700aaaatctatc aaaattctta tatatctttt tcgaatttga agtgaaattt cgataattta 8760aaattaaata gaacatatca ttatttaggt atcatattga tttttatact taattactaa 8820atttggttaa ctttgaaagt gtacatcaac gaaaaattag tcaaacgact aaaataaata 8880aatatcatgt gttattaaga aaattctcct ataagaatat tttaatagat catatgtttg 8940taaaaaaaat taatttttac taacacatat atttacttat caaaaatttg acaaagtaag 9000attaaaataa tattcatcta acaaaaaaaa aaccagaaaa tgctgaaaac ccggcaaaac 9060cgaaccaatc caaaccgata tagttggttt ggtttgattt tgatataaac cgaaccaact 9120cggtccattt gcacccctaa tcataatagc tttaatattt caagatatta ttaagttaac 9180gttgtcaata tcctggaaat tttgcaaaat gaatcaagcc tatatggctg taatatgaat 9240ttaaaagcag ctcgatgtgg tggtaatatg taatttactt gattctaaaa aaatatccca 9300agtattaata atttctgcta ggaagaaggt tagctacgat ttacagcaaa gccagaatac 9360aaagaaccat aaagtgattg aagctcgaaa tatacgaagg aacaaatatt tttaaaaaaa 9420tacgcaatga cttggaacaa aagaaagtga tatatttttt gttcttaaac aagcatcccc 9480tctaaagaat ggcagttttc ctttgcatgt aactattatg ctcccttcgt tacaaaaatt 9540ttggactact attgggaact tcttctgaaa atagtgatag aacccacacg agcatgtgct 9600ttccatttaa ttttaaaaac caagaaacat acatacataa cattccatca gcctctctct 9660ctttttatta cggttaatga cttaaaacac atcttattat cccatcctta acacctagca 9720gtgtctttat acgatctcat cgatcaccac ttcaaaacca tgcagactgc tgctgcccct 9780ggagctggca tcggctaggc tgggtgccgc actgtcccgg aaggtcccta gcgacttgtt 9840tagattgatg ggaccacctc tcaacttcct gctgctgtcc ctgctgctgg atgtcctgcc 9900tcatctggcc gattgcacgc tccagtcccc tgcatgtgca ctcgctcctc aattgcttaa 9960gatcatcgca gcagctatcg aagtgctggc tctgttgccc tcctccacgg ccttggttgt 10020agtagtagct gccgccgccc ttctggactt tttcccacag gaaccgccga ataattcgat 10080agaaccacac gagcatgtgc tttcatttat tttaaaaacc aagaaacata cataacattt 10140catcagcctc tctctctctc tctctctctc tctctctctc tctctctctc tctctctctt 10200tattacagct gttacactaa cttaaaacac attcatctca ttattattat tattatccat 10260ccttaacacc tagcagtgtc tttgtacgat ctcataatcg atcacccctt catcaggtat 10320ccttaggctt cactccaacg ttgttgcagt tacggaacat gtacacacca tcatggttct 10380caacgaactg gcaagatctc caagttttcc aaaggctaac ccacatgttc tcatcggtgt 10440gtctgtagtg ctctcccata actttcttga tgcactcggt agcttctcta gcatggtaga 10500atgggatcct tgaaacgtag tgatggagca catgagtctc gatgatgtca tggaagatga 10560ttccgaggat tccgaactct ctatcgatag tagcagcagc acccttagcg aaagtccact 10620cttgagcatc gtaatgaggc atagaagaat cggtgtgctg aaggaaggta acgaaaacaa 10680gccagtggtt aacaaggatc caaggacaga accatgtgat gaaagtaggc cagaatccga 10740aaaccttgta agcggtgtaa acagaagtga gggtagcaag gattccaaga tcagaaagaa 10800cgatgtacca gtagtccttc ttatcgaaaa cagggctaga aggccagtag tgagacttga 10860agaacttaga aacaccaggg taaggttgtc cagtagcgtt agtagcaagg taaagagaaa 10920gtcctccaag ctgttggaac aagagagcga aaacagagta gataggagtt tcctcagcga 10980tatcgtgaag gctggtaact tggtgcttct ctttgaattc ctcggcggtg taaggaacga 11040aaaccatatc tctggtcatg tgtccagtag ccttatggtg cttagcatga gagaacttcc 11100agctgaagta aggaaccata acaagagagt ggagaaccca tccaacggta tcgttaaccc 11160atccgtagtt agagaaagca gaatgtccac actcatgtcc aaggatccag attccgaatc 11220cgaaacaaga gatagagaac acgtaagcag accaagcagc gaatctaagg aattcgttag 11280ggagaagagg gatgtaggta agtccaacgt aagcgatagc agagatagcc acgatatctc 11340tcaccacgta agacatagac ttcacgagag atctctcgta acagtgctta gggatagcgt 11400caaggatatc cttgatggtg taatctggca ccttgaaaac gtttccgaag gtatcgatag 11460cggtcttttg ctgcttgaaa gatgcaacgt ttccagaacg cctaacggtc ttagtagatc 11520cctcaaggat ctcagatcca gacacggtaa ccttagacat ggtatggtaa ttgtaaatgt 11580aattgtaatg ttgtttgttg tttgttgttg ttggtaattg ttgtaaaatt tttggtggtg 11640attggttctt taaggtgtga gagtgagttg tgagttgtgt ggtgggtttg gtgagattgg 11700ggatggtggg tttatatagt ggagactgag gaatggggtc gtgagtgtta actttgcatg 11760ggctacacgt gggttctttt gggcttacac gtagtattat tcatgcaaat gcagccaata 11820catatacggt attttaataa tgtgtgggaa tacaatatgc cgagtatttt actaattttg 11880gcaatgacaa gtgtacattt ggattatctt acttggcctc tcttgcttta atttggatta 11940tttttattct cttaccttgg ccgttcatat tcacatccct aaaggcaaga cagaattgaa 12000tggtggccaa aaattaaaac gatggatatg acctacatag tgtaggatca attaacgtcg 12060aaggaaaata ctgattctct caagcatacg gacaagggta aataacatag tcaccagaac 12120ataataaaca aaaagtgcag aagcaagact aaaaaaatta gctatggaca ttcaggttca 12180tattggaaac atcattatcc tagtcttgtg accatccttc ctcctgctct agttgagagg 12240ccttgggact aacgagaggt cagttgggat agcagatcct tatcctggac tagcctttct 12300ggtgtttcag agtcttcgtg ccgccgtcta catctatctc cattaggtct gaagatgact 12360cttcacacca acgacgttta aggtctctat cctactccta gcttgcaata cctggcttgc 12420aatacctgga gcatcgtgca cgatgattgg atactgtgga ggaggagtgt ttgctgattt 12480agagctcccg gttgggtgat ttgacttcga tttcagttta ggcttgttga aatttttcag 12540gttccattgt gaagccttta gagcttgagc ttccttccat gttaatgcct tgatcgaata 12600ctcctagaga aaagggaagt cgatctctga gtattgaaat cgaagtgcac attttttttc 12660aacgtgtcca atcaatccac aaacaaagca gaagacaggt aatctttcat acttatactg 12720acaagtaata gtcttaccgt catgcataat aacgtctcgt tccttcaaga ggggttttcc 12780gacatccata acgacccgaa gcctcatgaa agcattaggg aagaactttt ggttcttctt 12840gtcatggcct ttataggtgt cagccgagct cgccaattcc cgtccgactg gctccgcaaa 12900atattcgaac ggcaagttat ggacttgcaa ccataactcc acggtattga gcaggaccta 12960ttgtgaagac tcatctcatg gagcttcaga atgtggttgt cagcaaacca atgaccgaaa 13020tccatcacat gacggacgtc cagtgggtga gcgaaacgaa acaggaagcg cctatctttc 13080agagtcgtga gctccacacc ggattccggc aactacgtgt tgggcaggct tcgccgtatt 13140agagatatgt tgaggcagac ccatctgtgc cactcgtaca attacgagag ttgttttttt 13200tgtgattttc ctagtttctc gttgatggtg agctcatatt ctacatcgta tggtctctca 13260acgtcgtttc ctgtcatctg atatcccgtc atttgcatcc acgtgcgccg cctcccgtgc 13320caagtcccta ggtgtcatgc acgccaaatt ggtggtggtg cgggctgccc tgtgcttctt 13380accgatgggt ggaggttgag tttgggggtc tccgcggcga tggtagtggg ttgacggttt 13440ggtgtgggtt gacggcattg atcaatttac ttcttgcttc aaattctttg gcagaaaaca 13500attcattaga ttagaactgg aaaccagagt gatgagacgg attaagtcag attccaacag 13560agttacatct cttaagaaat aatgtaaccc ctttagactt tatatatttg caattaaaaa 13620aataatttaa cttttagact ttatatatag ttttaataac taagtttaac cactctatta 13680tttatatcga aactatttgt atgtctcccc tctaaataaa cttggtattg tgtttacaga 13740acctataatc aaataatcaa tactcaactg aagtttgtgc agttaattga agggattaac 13800ggccaaaatg cactagtatt atcaaccgaa tagattcaca ctagatggcc atttccatca 13860atatcatcgc cgttcttctt ctgtccacat atcccctctg aaacttgaga gacacctgca 13920cttcattgtc cttattacgt gttacaaaat gaaacccatg catccatgca aactgaagaa 13980tggcgcaaga acccttcccc tccatttctt atgtggcgac catccatttc accatctccc 14040gctataaaac acccccatca cttcacctag aacatcatca ctacttgctt atccatccaa 14100aagataccca cttttacaac aattaccaac aacaacaaac aacaaacaac attacaatta 14160catttacaat taccatacca tgccacctag cgctgctaag caaatgggag cttctactgg 14220tgttcatgct ggtgttactg actcttctgc tttcaccaga aaggatgttg ctgatagacc 14280tgatctcacc atcgttggag attctgttta cgatgctaag gctttcagat ctgagcatcc 14340tggtggtgct catttcgttt ctttgttcgg aggaagagat gctactgagg ctttcatgga 14400ataccataga agggcttggc ctaagtctag aatgtctaga ttccacgttg gatctcttgc 14460ttctactgag gaacctgttg ctgctgatga gggatacctt caactttgtg ctaggatcgc 14520taagatggtg ccttctgttt cttctggatt cgctcctgct tcttactggg ttaaggctgg 14580acttatcctt ggatctgcta tcgctcttga ggcttacatg ctttacgctg gaaagagact 14640tctcccttct atcgttcttg gatggctttt cgctcttatc ggtcttaaca tccagcatga 14700tgctaaccat ggtgctttgt ctaagtctgc ttctgttaac cttgctcttg gactttgtca 14760ggattggatc ggaggatcta tgatcctttg gcttcaagag catgttgtta tgcaccacct 14820ccacactaac gatgttgata aggatcctga tcaaaaggct cacggtgctc ttagactcaa 14880gcctactgat gcttggtcac ctatgcattg gcttcagcat ctttaccttt tgcctggtga 14940gactatgtac gctttcaagc ttttgttcct cgacatctct gagcttgtta tgtggcgttg 15000ggagggtgag cctatctcta agcttgctgg atacctcttt atgccttctt tgcttctcaa 15060gcttaccttc tgggctagat tcgttgcttt gcctctttac cttgctcctt ctgttcatac 15120tgctgtgtgt atcgctgcta ctgttatgac tggatctttc tacctcgctt tcttcttctt 15180catctcccac aacttcgagg gtgttgcttc tgttggacct gatggatcta tcacttctat 15240gactagaggt gctagcttcc ttaagagaca agctgagact tcttctaacg ttggaggacc 15300tcttcttgct actcttaacg gtggactcaa ctaccaaatt gagcatcact tgttccctag 15360agttcaccat ggattctacc ctagacttgc tcctcttgtt aaggctgagc ttgaggctag 15420aggaatcgag tacaagcact accctactat ctggtctaac cttgcttcta ccctcagaca 15480tatgtacgct cttggaagaa ggcctagatc taaggctgag taatgacaag cttatgtgac 15540gtgaaataat aacggtaaaa tatatgtaat aataataata ataaagccac aaagtgagaa 15600tgaggggaag gggaaatgtg taatgagcca gtagccggtg gtgctaattt tgtatcgtat 15660tgtcaataaa tcatgaattt tgtggttttt atgtgttttt ttaaatcatg aattttaaat 15720tttataaaat aatctccaat cggaagaaca acattccata tccatgcatg gatgtttctt 15780tacccaaatc tagttcttga gaggatgaag catcaccgaa cagttctgca actatccctc 15840aaaagcttta aaatgaacaa caaggaacag agcaacgttc caaagatccc aaacgaaaca 15900tattatctat actaatacta tattattaat tactactgcc cggaatcaca atccctgaat 15960gattcctatt aactacaagc cttgttggcg gcggagaagt gatcggcgcg gcgagaagca 16020gcggactcgg agacgaggcc ttggaagatc tgagtcgaac gggcagaatc agtattttcc 16080ttcgacgtta attgatccta cactatgtag gtcatatcca tcgttttaat ttttggccac 16140cattcaattc tgtcttgcct ttagggatgt gaatatgaac ggccaaggta agagaataaa 16200aataatccaa attaaagcaa gagaggccaa gtaagataat ccaaatgtac acttgtcatt 16260gccaaaatta gtaaaatact cggcatattg tattcccaca cattattaaa ataccgtata 16320tgtattggct gcatttgcat gaataatact acgtgtaagc ccaaaagaac ccacgtgtag 16380cccatgcaaa gttaacactc acgaccccat tcctcagtct ccactatata aacccaccat 16440ccccaatctc accaaaccca ccacacaact cacaactcac tctcacacct taaagaacca 16500atcaccacca aaaattttac aacaattacc aacaacaaca aacaacaaac aacattacaa 16560ttacatttac aattaccata ccatgagcgc tgttaccgtt actggatctg atcctaagaa 16620cagaggatct tctagcaaca ccgagcaaga ggttccaaaa gttgctatcg ataccaacgg 16680aaacgtgttc tctgttcctg atttcaccat caaggacatc cttggagcta tccctcatga 16740gtgttacgag agaagattgg ctacctctct ctactacgtg ttcagagata tcttctgcat 16800gcttaccacc ggatacctta cccataagat cctttaccct ctcctcatct cttacacctc 16860taacagcatc atcaagttca ctttctgggc cctttacact tacgttcaag gacttttcgg 16920aaccggaatc tgggttctcg ctcatgagtg tggacatcaa gctttctctg attacggaat 16980cgtgaacgat ttcgttggat ggacccttca ctcttacctt atggttcctt acttcagctg 17040gaagtactct catggaaagc accataaggc tactggacac atgaccagag atatggtttt 17100cgttcctgcc accaaagagg aattcaagaa gtctaggaac ttcttcggta acctcgctga 17160gtactctgag gattctccac ttagaaccct ttacgagctt cttgttcaac aacttggagg 17220atggatcgct tacctcttcg ttaacgttac aggacaacct taccctgatg ttccttcttg 17280gaaatggaac cacttctggc ttacctctcc acttttcgag caaagagatg ctctctacat 17340cttcctttct gatcttggaa tcctcaccca gggaatcgtt cttactcttt ggtacaagaa 17400attcggagga tggtcccttt tcatcaactg gttcgttcct tacatctggg ttaaccactg 17460gctcgttttc atcacattcc ttcagcacac tgatcctact atgcctcatt acaacgctga 17520ggaatggact ttcgctaagg gtgctgctgc tactatcgat agaaagttcg gattcatcgg 17580acctcacatc ttccatgata tcatcgagac tcatgtgctt caccactact gttctaggat 17640cccattctac aacgctagac ctgcttctga ggctatcaag aaagttatgg gaaagcacta 17700caggtctagc gacgagaaca tgtggaagtc actttggaag tctttcaggt cttgccaata 17760cgttgacggt gataacggtg ttctcatgtt ccgtaacatc aacaactgcg gagttggagc 17820tgctgagaag taatgaaggg gtgatcgatt atgagatcgt acaaagacac tgctaggtgt 17880taaggatgga taataataat aataatgaga tgaatgtgtt ttaagttagt gtaacagctg 17940taataaagag agagagagag agagagagag agagagagag agagagagag agagagaggc 18000tgatgaaatg ttatgtatgt ttcttggttt ttaaaataaa tgaaagcaca tgctcgtgtg 18060gttctatcga attattcggc ggttcctgtg ggaaaaagtc cagaagggcc gccgcagcta 18120ctactacaac caaggccgtg gaggagggca acagagccag cacttcgata gctgctgcga 18180tgatcttaag caattgagga gcgagtgcac atgcagggga ctggagcgtg caatcggcca 18240gatgaggcag gacatccagc agcagggaca gcagcaggaa gttgagaggt ggtcccatca 18300atctaaacaa gtcgctaggg accttccggg acagtgcggc acccagccta gccgatgcca 18360gctccagggg cagcagcagt ctgcatggtt ttgaagtggt gatcgatgag atcgtataaa 18420gacactgcta ggtgttaagg atgggataat aagatgtgtt ttaagtcatt aaccgtaata 18480aaaagagaga gaggctgatg gaatgttatg tatgtatgtt tcttggtttt taaaattaaa 18540tggaaagcac atgctcgtgt gggttctatc tcgattaaaa atcccaatta tatttggtct 18600aatttagttt ggtattgagt aaaacaaatt cgaaccaaac caaaatataa atatatagtt 18660tttatatata tgcctttaag actttttata gaattttctt taaaaaatat ctagaaatat 18720ttgcgactct tctggcatgt aatatttcgt taaatatgaa gtgctccatt tttattaact 18780ttaaataatt ggttgtacga tcactttctt atcaagtgtt actaaaatgc gtcaatctct 18840ttgttcttcc atattcatat gtcaaaatct atcaaaattc ttatatatct ttttcgaatt 18900tgaagtgaaa tttcgataat ttaaaattaa atagaacata tcattattta ggtatcatat 18960tgatttttat acttaattac taaatttggt taactttgaa agtgtacatc aacgaaaaat 19020tagtcaaacg actaaaataa ataaatatca tgtgttatta agaaaattct cctataagaa 19080tattttaata gatcatatgt ttgtaaaaaa aattaatttt tactaacaca tatatttact 19140tatcaaaaat ttgacaaagt aagattaaaa taatattcat ctaacaaaaa aaaaaccaga 19200aaatgctgaa aacccggcaa aaccgaacca atccaaaccg atatagttgg tttggtttga 19260ttttgatata aaccgaacca actcggtcca tttgcacccc taatcataat agctttaata 19320tttcaagata ttattaagtt aacgttgtca atatcctgga aattttgcaa aatgaatcaa 19380gcctatatgg ctgtaatatg aatttaaaag cagctcgatg tggtggtaat atgtaattta 19440cttgattcta aaaaaatatc ccaagtatta ataatttctg ctaggaagaa ggttagctac 19500gatttacagc aaagccagaa tacaaagaac cataaagtga ttgaagctcg aaatatacga 19560aggaacaaat atttttaaaa aaatacgcaa tgacttggaa caaaagaaag tgatatattt 19620tttgttctta aacaagcatc ccctctaaag aatggcagtt ttcctttgca tgtaactatt 19680atgctccctt cgttacaaaa attttggact actattggga acttcttctg aaaatagtcc 19740tgcaggctag tagattggtt ggttggtttc catgtaccag aaggcttacc ctattagttg 19800aaagttgaaa ctttgttccc tactcaattc ctagttgtgt aaatgtatgt atatgtaatg 19860tgtataaaac gtagtactta aatgactagg agtggttctt gagaccgatg agagatggga 19920gcagaactaa agatgatgac ataattaaga acgaatttga aaggctctta ggtttgaatc 19980ctattcgaga atgtttttgt caaagatagt ggcgattttg aaccaaagaa aacatttaaa 20040aaatcagtat ccggttacgt tcatgcaaat agaaagtggt ctaggatctg attgtaattt 20100tagacttaaa gagtctctta agattcaatc ctggctgtgt acaaaactac aaataatata 20160ttttagacta tttggcctta actaaacttc cactcattat ttactgaggt tagagaatag 20220acttgcgaat aaacacattc ccgagaaata ctcatgatcc cataattagt cagagggtat 20280gccaatcaga tctaagaaca cacattccct caaattttaa tgcacatgta atcatagttt 20340agcacaattc aaaaataatg tagtattaaa gacagaaatt tgtagacttt tttttggcgt 20400taaaagaaga ctaagtttat acgtacattt tattttaagt ggaaaaccga aattttccat 20460cgaaatatat gaatttagta tatatatttc tgcaatgtac tattttgcta ttttggcaac 20520tttcagtgga ctactacttt attacaatgt gtatggatgc atgagtttga gtatacacat 20580gtctaaatgc atgctttgta aaacgtaacg gaccacaaaa gaggatccat acaaatacat 20640ctcatagctt cctccattat tttccgacac aaacagagca ttttacaaca attaccaaca 20700acaacaaaca acaaacaaca ttacaattac atttacaatt accataccat ggaatttgct 20760caacctctcg ttgctatggc tcaagagcag tacgctgcta tcgatgctgt tgttgctcct 20820gctatcttct ctgctaccga ctctattgga tggggactca agcctatctc ttctgctact 20880aaggatctcc ctctcgttga atctcctacc cctcttatcc tttctctcct cgcttacttc 20940gctatcgttg gttctggact cgtttaccgt aaagtgttcc ctagaaccgt taagggacag 21000gatcctttcc ttctcaaggc tcttatgctc gctcacaacg ttttccttat cggactcagc 21060ctttacatgt gcctcaagct cgtttacgag gcttacgtga acaagtactc cttctgggga 21120aacgcttaca accctgctca aaccgagatg gctaaggtga tctggatctt ctacgtgtcc 21180aagatctacg agttcatgga caccttcatc atgcttctca agggaaacgt taaccaggtt 21240tccttcctcc atgtttacca ccacggatct atctctggaa tctggtggat gatcacttat 21300gctgctccag gtggagatgc ttacttctct gctgctctca actcttgggt tcatgtgtgc 21360atgtacacct actacttcat ggctgctgtt cttcctaagg acgaaaagac caagagaaag 21420tacctttggt ggggaagata ccttacccag atgcaaatgt tccagttctt catgaacctt 21480ctccaggctg tttacctcct ctactcttct tctccttacc ctaagttcat tgctcaactc 21540ctcgttgttt acatggttac cctcctcatg cttttcggaa acttctacta catgaagcac 21600cacgcttcta agtgataagg gccgccgcca tgtgacagat cgaaggaaga aagtgtaata 21660agacgactct cactactcga tcgctagtga ttgtcattgt tatatataat aatgttatct 21720ttcacaactt atcgtaatgc atgtgaaact ataacacatt aatcctactt gtcatatgat 21780aacactctcc ccatttaaaa ctcttgtcaa tttaaagata taagattctt taaatgatta 21840aaaaaaatat attataaatt caatcactcc tactaataaa ttattaatta ttatttattg 21900attaaaaaaa tacttatact aatttagtct gaatagaata attagattct agcctgcagg 21960gcggccgcgg atcccatgga gtcaaagatt caaatagagg acctaacaga actcgccgta 22020aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa gaaaatcttc 22080gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga tacagtctca 22140gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa cctcctcgga 22200ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga aggtggctcc 22260tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc tgccgacagt 22320ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc 22380acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa 22440tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagaga 22500acacggggga ctgaattaaa tatgagccct gagaggcgtc ctgttgaaat cagacctgct 22560actgctgctg atatggctgc tgtttgtgat atcgtgaacc actacatcga gacttctacc 22620gttaacttca gaactgagcc tcaaactcct caagagtgga tcgatgatct tgagagactc 22680caagatagat acccttggct tgttgctgag gttgagggtg ttgttgctgg aatcgcttac 22740gctggacctt ggaaggctag aaacgcttac gattggactg ttgagtctac cgtttacgtt 22800tcacacagac atcagagact tggacttgga tctacccttt acactcacct tctcaagtct 22860atggaagctc agggattcaa gtctgttgtt gctgttatcg gactccctaa cgatccttct 22920gttagacttc atgaggctct tggatacact gctagaggaa ctcttagagc tgctggatac 22980aagcacggtg gatggcatga tgttggattc tggcaaagag atttcgagct tcctgctcct 23040cctagacctg ttagaccagt tactcagatc tgaatttgcg tgatcgttca aacatttggc 23100aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc 23160tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat 23220gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat 23280agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcactagt 23340gatgtacggt taaaaccacc ccagtacatt aaaaacgtcc gcaatgtgtt attaagttgt 23400ctaagcgtca atttgtttac accacaatat atcctgccac cagccagcca acagctcccc 23460gaccggcagc tcggcacaaa atcaccactc gatacaggca gcccatcagt cc

23512325787DNAArtificial SequencepGA7- mod_C nucleotide sequence 3tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180tcacgacgtt gtaaaacggg cgccccgatc tagtaacata gatgacaccg cgcgcgataa 240tttatcctag tttgcgcgct atattttgtt ttctatcgcg tattaaatgt ataattgcgg 300gactctaatc ataaaaaccc atctcataaa taacgtcatg cattacatgt taattattac 360gtgcttaacg taattcaaca gaaattatat gataatcatc gcaagaccgg caacaggatt 420caatcttaag aaactttatt gccaaatgtt tgaacgatct gcccggaagc ggccaactcg 480aaaatttaat taatcatcag tgagccttct cagcctttcc gttaacgtag tagtgctgtc 540caactttgtc gaggttgctg aaagtagcct tccaagcacc gtagtaagag agcaccttgt 600agttgagtcc ccacttctta gcgaaaggga cgaatcttct tgacacctca ggctgtctga 660attgaggcat atcagggaag agatggtgga taacctggca gttaaggtat cccataagcc 720agttaacgta tccacgagaa ggatcgatgt caacggtgtg atcaacagcg tagttaaccc 780agctaaggtg cttgtcagat ggaacaacag ggaggtgagt gtgagaagta gagaagtgag 840cgaagaggta catgtaagcg atccagtttc cgaaagtgaa ccaccagtaa gcaacaggcc 900aagagtatcc ggtagcaagc ttgataacag cggttctaac aacgtgagaa acgagcatcc 960aagaagcttc ctcgtagttc ttcttcctga gcacctgtct aggatggaga acgtagatcc 1020agaaagcctg aacgagaagt ccagaagtaa caggaacgaa ggtccaagct tgaagtctag 1080cccaagctct agagaatccc ctaggtctat tatcctccac agcggtgttg aagaaagcca 1140cagcaggagt ggtatcaaga tccatgtcgt gtctaacttt ctgaggggta gcatggtgct 1200tgttatgcat ctggttccac atctctccgc tggtagaaag tccgaatccg caagtcatag 1260cctgaagtct cttatccacg tacacagatc cggtaagaga gttgtgtcca ccctcatgtt 1320gaacccatcc acatctagct ccgaagaaag caccgtacac aacgctagca atgatagggt 1380atccagcgta cataagagcg gttccaagag cgaaagtagc aagaagctcc aaaagacggt 1440aagcaacatg ggtgatagaa ggcttgaaga atccgtccct ctcaagttca gctctccacc 1500tagcgaaatc ctcaagcata ggagcatcct cagactcaga tctcttgatc tcagcaggtc 1560tagaaggcaa agctctaagc atcttccaag ccttgaggct acgcatgtga aattctttga 1620aagcctcagt agcatcagca ccagtgttag caagcatgta gaagatcacg cttccaccag 1680gatgtttgaa gttggtcacg tcgtactcaa catcctcaac cctaacccat ctagtctcga 1740aggtagcagc aagttcatga ggctcaaggg tcttaagatc aacaggagcg gtagaagcat 1800ccttagcatc aagagcctca gcagatgact tagacctggt gagaggagat ctaggagaag 1860atcttccatc ggtcttagga ggacacatgg cgcgccgatt ttcgagatgg taattgtaaa 1920tgtaattgta atgttgtttg ttgtttgttg ttgttggtaa ttgttgtaaa attcgagttg 1980gccgcttccg gggatcctcg tgtatgtttt taatcttgtt tgtatcgatg agttttggtt 2040tgagtaaaga gtgaagcgga tgagttaatt tataggctat aaaggagatt tgcatggcga 2100tcacgtgtaa taatgcatgc acgcatgtga ttgtatgtgt gtgctgtgag agagaagctc 2160ttaggtgttt gaagggagtg acaagtggcg aagaaaaaca attctccgcg gctgcatgct 2220atgtgtaacg tgtagctaat gttctggcat ggcatcttat gaacgattct ttttaaaaac 2280aaggtaaaaa cttaacttca taaaattaaa aaaaaaaacg tttactaagt tggtttaaaa 2340ggggatgaga ggcgccccgc ggaaagcttg ctagccaatt ggggcccaac gttctcgagt 2400ttttctagaa ggaaactgaa ggcgggaaac gacaatctgc tagtggatct cccagtcacg 2460acgttgtaaa acgggcgccc cgcggaaagc ttgcggccgc ggtaccgccc gttcgactca 2520gatcttccaa ggcctcgtct ccgagtccgc tgcttctcgc cgcgccgatc acttctccgc 2580cgccaacaag gcttgtagtt aataggaatc attcagggat tgtgattccg ggcagtagta 2640attaataata tagtattagt atagataata tgtttcgttt gggatctttg gaacgttgct 2700ctgttccttg ttgttcattt taaagctttt gagggatagt tgcagaactg ttcggtgatg 2760cttcatcctc tcaagaacta gatttgggta aagaaacatc catgcatgga tatggaatgt 2820tgttcttccg attggagatt attttataaa atttaaaatt catgatttaa aaaaacacat 2880aaaaaccaca aaattcatga tttattgaca atacgataca aaattagcac caccggctac 2940tggctcatta cacatttccc cttcccctca ttctcacttt gtggctttat tattattatt 3000attacatata ttttaccgtt attatttcac gtcacataag cttgttaatt aatcattagt 3060gagccttctc agcctttccg ttaacgtagt agtgctgtcc caccttatca aggttagaga 3120aagtagcctt ccaagcaccg tagtaagaga gcaccttgta gttgagtccc cacttcttag 3180cgaaaggaac gaatcttctg ctaacctcag gctgtctgaa ttgaggcata tcagggaaga 3240ggtggtggat aacctgacag ttaaggtatc ccataagcca gttcacgtat cctctagaag 3300gatcgatatc aacggtgtga tcaacagcgt agttaaccca agaaaggtgc ttatcagatg 3360gaacaacagg gaggtgagta tgagaagtag agaagtgagc gaaaaggtac atgtaagcga 3420tccagtttcc gaaagtgaac caccagtaag caacaggcca agagtatcca gtagcaagct 3480tgataacagc ggttctaaca acatgagaaa cgagcatcca agaagcctct tcgtagttct 3540tcttacggag aacttgtcta gggtggagaa cgtagatcca gaaagcttga acaagaagtc 3600cagaggtaac aggaacgaaa gtccaagctt gaagtctagc ccaagctcta gagaatcctc 3660taggtctgtt atcctcaaca gcagtgttga agaaagccac agcaggagtg gtatcaagat 3720ccatatcgtg tctaaccttt tgaggggtag catggtgctt gttatgcatc tggttccaca 3780tctcaccaga agtagaaagt ccgaatccac aagtcatagc ctgaagtctc ttgtccacgt 3840aaacagatcc ggtaagagag ttatgtccac cctcatgttg aacccatcca catctagctc 3900cgaagaaagc accgtaaaca acagaagcaa tgatagggta tccagcgtac ataagagcag 3960ttccaagagc gaatgtagca agaagctcga gaagtctgta agccacatgg gtgatagaag 4020gcttgaagaa tccatctctc tcaagctcag cacgccatct agcgaaatcc tcaagcatag 4080gagcatcctc agactcagat ctcttgatct cagcaggtct agaaggcaaa gctctaagca 4140tcttccaagc cttgagagaa cgcatgtgga attctttgaa agcctcagta gcatcagcac 4200cagtgttagc aagcatgtag aagatcacag atccaccagg gtgcttgaag ttagtcacat 4260cgtactcaac gtcctcaact ctaacccatc tagtctcgaa agtagcagca agctcatgag 4320gctcaagagt cttaagatca acaggagcag tagaagcatc cttagcatca agagcctcag 4380cagaagattt agacctggta agtggagatc taggagaaga tcttccatca gtcttaggag 4440ggcacatggt atggtaattg taaatgtaat tgtaatgttg tttgttgttt gttgttgttg 4500gtaattgttg taaaattaat taagtgggta tcttttggat ggataagcaa gtagtgatga 4560tgttctaggt gaagtgatgg gggtgtttta tagcgggaga tggtgaaatg gatggtcgcc 4620acataagaaa tggaggggaa gggttcttgc gccattcttc agtttgcatg gatgcatggg 4680tttcattttg taacacgtaa taaggacaat gaagtgcagg tgtctctcaa gtttcagagg 4740ggatatgtgg acagaagaag aacggcgatg atattgatgg aaatggccat ctagtgtgaa 4800tctattcggt tgataatact agtgcatttt ggccgttaat cccttcaatt aactgcacaa 4860acttcagttg agtattgatt atttgattat aggttctgta aacacaatac caagtttatt 4920tagaggggag acatacaaat agtttcgata taaataatag agtggttaaa cttagttatt 4980aaaactatat ataaagtcta aaagttaaat tattttttta attgcaaata tataaagtct 5040aaaggggtta cattatttct taagagatgt aactctgttg gaatctgact taatccgtct 5100catcactctg gtttccagtt ctaatctaat gaattgtttt ctgccaaaga atttgaagca 5160agaagtaaat tgatcaatgc cgtcaaccca caccaaaccg tcaacccact accatcgccg 5220cggagacccc caaactcaac ctccacccat cggtaagaag cacagggcag cccgcaccac 5280caccaatttg gcgtgcatga cacctaggga cttggcacgg gaggcggcgc acgtggatgc 5340aaatgacggg atatcagatg acaggaaacg acgttgagag accatacgat gtagaatatg 5400agctcaccat caacgagaaa ctaggaaaat cacaaaaaaa acaactctcg taattgtacg 5460agtggcacag atgggtctgc ctcaacatat ctctaatacg gcgaagcctg cccaacacgt 5520agttgccgga atccggtgtg gagctcacga ctctgaaaga taggcgcttc ctgtttcgtt 5580tcgctcaccc actggacgtc cgtcatgtga tggatttcgg tcattggttt gctgacaacc 5640acattctgaa gctccatgag atgagtcttc acaataggtc ctgctcaata ccgtggagtt 5700atggttgcaa gtccataact tgccgttcga atattttgcg gagccagtcg gacgggaatt 5760ggcgagctcg gctgacacct ataaaggcca tgacaagaag aaccaaaagt tcttccctaa 5820tgctttcatg aggcttcggg tcgttatgga tgtcggaaaa cccctcttga aggaacgaga 5880cgttattatg catgacggta agactattac ttgtcagtat aagtatgaaa gattacctgt 5940cttctgcttt gtttgtggat tgattggaca cgttgaaaaa aaatgtgcac ttcgatttca 6000atactcagag atcgacttcc cttttctcta ggagtattcg atcaaggcat taacatggaa 6060ggaagctcaa gctctaaagg cttcacaatg gaacctgaaa aatttcaaca agcctaaact 6120gaaatcgaag tcaaatcacc caaccgggag ctctaaatca gcaaacactc ctcctccaca 6180gtatccaatc atcgtgcacg atgctccagg tattgcaagc caggtattgc aagctaggag 6240taggatagag accttaaacg tcgttggtgt gaagagtcat cttcagacct aatggagata 6300gatgtagacg gcggcacgaa gactctgaaa caccagaaag gctagtccag gataaggatc 6360tgctatccca actgacctct cgttagtccc aaggcctctc aactagagca ggaggaagga 6420tggtcacaag actaggataa tgatgtttcc aatatgaacc tgaatgtcca tagctaattt 6480ttttagtctt gcttctgcac tttttgttta ttatgttctg gtgactatgt tatttaccct 6540tgtccgtatg cttgagggta ccctagtaga ttggttggtt ggtttccatg taccagaagg 6600cttaccctat tagttgaaag ttgaaacttt gttccctact caattcctag ttgtgtaaat 6660gtatgtatat gtaatgtgta taaaacgtag tacttaaatg actaggagtg gttcttgaga 6720ccgatgagag atgggagcag aactaaagat gatgacataa ttaagaacga atttgaaagg 6780ctcttaggtt tgaatcctat tcgagaatgt ttttgtcaaa gatagtggcg attttgaacc 6840aaagaaaaca tttaaaaaat cagtatccgg ttacgttcat gcaaatagaa agtggtctag 6900gatctgattg taattttaga cttaaagagt ctcttaagat tcaatcctgg ctgtgtacaa 6960aactacaaat aatatatttt agactatttg gccttaacta aacttccact cattatttac 7020tgaggttaga gaatagactt gcgaataaac acattcccga gaaatactca tgatcccata 7080attagtcaga gggtatgcca atcagatcta agaacacaca ttccctcaaa ttttaatgca 7140catgtaatca tagtttagca caattcaaaa ataatgtagt attaaagaca gaaatttgta 7200gacttttttt tggcgttaaa agaagactaa gtttatacgt acattttatt ttaagtggaa 7260aaccgaaatt ttccatcgaa atatatgaat ttagtatata tatttctgca atgtactatt 7320ttgctatttt ggcaactttc agtggactac tactttatta caatgtgtat ggatgcatga 7380gtttgagtat acacatgtct aaatgcatgc tttgtaaaac gtaacggacc acaaaagagg 7440atccatacaa atacatctca tagcttcctc cattattttc cgacacaaac agagcatttt 7500acaacaatta ccaacaacaa caaacaacaa acaacattac aattacattt acaattacca 7560taccatggcc tctatcgcta tccctgctgc tcttgctgga actcttggat acgttaccta 7620caatgtggct aaccctgata tcccagcttc tgagaaagtt cctgcttact tcatgcaggt 7680tgagtactgg ggacctacta tcggaactat tggatacctc ctcttcatct acttcggaaa 7740gcgtatcatg cagaacagat ctcaaccttt cggactcaag aacgctatgc tcgtttacaa 7800cttctaccag accttcttca acagctactg catctacctt ttcgttactt ctcatagggc 7860tcagggactt aaggtttggg gaaacatccc tgatatgact gctaactctt ggggaatctc 7920tcaggttatc tggcttcact acaacaacaa gtacgttgag cttctcgaca ccttcttcat 7980ggtgatgagg aagaagttcg accagctttc tttccttcac atctaccacc acactcttct 8040catctggtca tggttcgttg ttatgaagct tgagcctgtt ggagattgct acttcggatc 8100ttctgttaac accttcgtgc acgtgatcat gtactcttac tacggacttg ctgctcttgg 8160agttaactgt ttctggaaga agtacatcac ccagatccag atgcttcagt tctgtatctg 8220tgcttctcac tctatctaca ccgcttacgt tcagaatacc gctttctggc ttccttacct 8280tcaactctgg gttatggtga acatgttcgt tctcttcgcc aacttctacc gtaagaggta 8340caagtctaag ggtgctaaga agcagtgata aggcgcgcgg cgcgccgggc cgccgccatg 8400tgacagatcg aaggaagaaa gtgtaataag acgactctca ctactcgatc gctagtgatt 8460gtcattgtta tatataataa tgttatcttt cacaacttat cgtaatgcat gtgaaactat 8520aacacattaa tcctacttgt catatgataa cactctcccc atttaaaact cttgtcaatt 8580taaagatata agattcttta aatgattaaa aaaaatatat tataaattca atcactccta 8640ctaataaatt attaattatt atttattgat taaaaaaata cttatactaa tttagtctga 8700atagaataat tagattctag tctcatcccc ttttaaacca acttagtaaa cgtttttttt 8760tttaatttta tgaagttaag tttttacctt gtttttaaaa agaatcgttc ataagatgcc 8820atgccagaac attagctaca cgttacacat agcatgcagc cgcggagaat tgtttttctt 8880cgccacttgt cactcccttc aaacacctaa gagcttctct ctcacagcac acacatacaa 8940tcacatgcgt gcatgcatta ttacacgtga tcgccatgca aatctccttt atagcctata 9000aattaactca tccgcttcac tctttactca aaccaaaact catcgataca aacaagatta 9060aaaacataca cgaggatctt ttacaacaat taccaacaac aacaaacaac aaacaacatt 9120acaattacat ttacaattac cataccatgc ctccaaggga ctcttactct tatgctgctc 9180ctccttctgc tcaacttcac gaagttgata ctcctcaaga gcacgacaag aaagagcttg 9240ttatcggaga tagggcttac gatgttacca acttcgttaa gagacaccct ggtggaaaga 9300tcattgctta ccaagttgga actgatgcta ccgatgctta caagcagttc catgttagat 9360ctgctaaggc tgacaagatg cttaagtctc ttccttctcg tcctgttcac aagggatact 9420ctccaagaag ggctgatctt atcgctgatt tccaagagtt caccaagcaa cttgaggctg 9480agggaatgtt cgagccttct cttcctcatg ttgcttacag acttgctgag gttatcgcta 9540tgcatgttgc tggtgctgct cttatctggc atggatacac tttcgctgga atcgctatgc 9600ttggagttgt tcagggaaga tgtggatggc ttatgcatga gggtggacat tactctctca 9660ctggaaacat tgctttcgac agagctatcc aagttgcttg ttacggactt ggatgtggaa 9720tgtctggtgc ttggtggcgt aaccagcata acaagcacca tgctactcct caaaagcttc 9780agcacgatgt tgatcttgat acccttcctc tcgttgcttt ccatgagaga atcgctgcta 9840aggttaagtc tcctgctatg aaggcttggc tttctatgca agctaagctt ttcgctcctg 9900ttaccactct tcttgttgct cttggatggc agctttacct tcatcctaga cacatgctca 9960ggactaagca ctacgatgag cttgctatgc tcggaatcag atacggactt gttggatacc 10020ttgctgctaa ctacggtgct ggatacgttc tcgcttgtta ccttctttac gttcagcttg 10080gagctatgta catcttctgc aacttcgctg tttctcatac tcacctccct gttgttgagc 10140ctaacgagca tgctacttgg gttgagtacg ctgctaacca cactactaac tgttctccat 10200cttggtggtg tgattggtgg atgtcttacc ttaactacca gatcgagcac cacctttacc 10260cttctatgcc tcaattcaga caccctaaga tcgctcctag agttaagcag cttttcgaga 10320agcacggact tcactacgat gttagaggat acttcgaggc tatggctgat actttcgcta 10380accttgataa cgttgcccat gctcctgaga agaaaatgca gtaatgagat cgttcaaaca 10440tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat 10500aatttctgtt gaattacgtt aagcacgtaa taattaacat gtaatgcatg acgttattta 10560tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg atagaaaaca 10620aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagatc 10680ggtcgattaa aaatcccaat tatatttggt ctaatttagt ttggtattga gtaaaacaaa 10740ttcgaaccaa accaaaatat aaatatatag tttttatata tatgccttta agacttttta 10800tagaattttc tttaaaaaat atctagaaat atttgcgact cttctggcat gtaatatttc 10860gttaaatatg aagtgctcca tttttattaa ctttaaataa ttggttgtac gatcactttc 10920ttatcaagtg ttactaaaat gcgtcaatct ctttgttctt ccatattcat atgtcaaaat 10980ctatcaaaat tcttatatat ctttttcgaa tttgaagtga aatttcgata atttaaaatt 11040aaatagaaca tatcattatt taggtatcat attgattttt atacttaatt actaaatttg 11100gttaactttg aaagtgtaca tcaacgaaaa attagtcaaa cgactaaaat aaataaatat 11160catgtgttat taagaaaatt ctcctataag aatattttaa tagatcatat gtttgtaaaa 11220aaaattaatt tttactaaca catatattta cttatcaaaa atttgacaaa gtaagattaa 11280aataatattc atctaacaaa aaaaaaacca gaaaatgctg aaaacccggc aaaaccgaac 11340caatccaaac cgatatagtt ggtttggttt gattttgata taaaccgaac caactcggtc 11400catttgcacc cctaatcata atagctttaa tatttcaaga tattattaag ttaacgttgt 11460caatatcctg gaaattttgc aaaatgaatc aagcctatat ggctgtaata tgaatttaaa 11520agcagctcga tgtggtggta atatgtaatt tacttgattc taaaaaaata tcccaagtat 11580taataatttc tgctaggaag aaggttagct acgatttaca gcaaagccag aatacaaaga 11640accataaagt gattgaagct cgaaatatac gaaggaacaa atatttttaa aaaaatacgc 11700aatgacttgg aacaaaagaa agtgatatat tttttgttct taaacaagca tcccctctaa 11760agaatggcag ttttcctttg catgtaacta ttatgctccc ttcgttacaa aaattttgga 11820ctactattgg gaacttcttc tgaaaatagt gatagaaccc acacgagcat gtgctttcca 11880tttaatttta aaaaccaaga aacatacata cataacattc catcagcctc tctctctttt 11940tattacggtt aatgacttaa aacacatctt attatcccat ccttaacacc tagcagtgtc 12000tttatacgat ctcatcgatc accacttcaa aaccatgcag actgctgctg cccctggagc 12060tggcatcggc taggctgggt gccgcactgt cccggaaggt ccctagcgac ttgtttagat 12120tgatgggacc acctctcaac ttcctgctgc tgtccctgct gctggatgtc ctgcctcatc 12180tggccgattg cacgctccag tcccctgcat gtgcactcgc tcctcaattg cttaagatca 12240tcgcagcagc tatcgaagtg ctggctctgt tgccctcctc cacggccttg gttgtagtag 12300tagctgccgc cgcccttctg gactttttcc cacaggaacc gccgaataat tcgatagaac 12360cacacgagca tgtgctttca tttattttaa aaaccaagaa acatacataa catttcatca 12420gcctctctct ctctctctct ctctctctct ctctctctct ctctctctct ctctttatta 12480cagctgttac actaacttaa aacacattca tctcattatt attattatta tccatcctta 12540acacctagca gtgtctttgt acgatctcat aatcgatcac cccttcatca ggtatcctta 12600ggcttcactc caacgttgtt gcagttacgg aacatgtaca caccatcatg gttctcaacg 12660aactggcaag atctccaagt tttccaaagg ctaacccaca tgttctcatc ggtgtgtctg 12720tagtgctctc ccataacttt cttgatgcac tcggtagctt ctctagcatg gtagaatggg 12780atccttgaaa cgtagtgatg gagcacatga gtctcgatga tgtcatggaa gatgattccg 12840aggattccga actctctatc gatagtagca gcagcaccct tagcgaaagt ccactcttga 12900gcatcgtaat gaggcataga agaatcggtg tgctgaagga aggtaacgaa aacaagccag 12960tggttaacaa ggatccaagg acagaaccat gtgatgaaag taggccagaa tccgaaaacc 13020ttgtaagcgg tgtaaacaga agtgagggta gcaaggattc caagatcaga aagaacgatg 13080taccagtagt ccttcttatc gaaaacaggg ctagaaggcc agtagtgaga cttgaagaac 13140ttagaaacac cagggtaagg ttgtccagta gcgttagtag caaggtaaag agaaagtcct 13200ccaagctgtt ggaacaagag agcgaaaaca gagtagatag gagtttcctc agcgatatcg 13260tgaaggctgg taacttggtg cttctctttg aattcctcgg cggtgtaagg aacgaaaacc 13320atatctctgg tcatgtgtcc agtagcctta tggtgcttag catgagagaa cttccagctg 13380aagtaaggaa ccataacaag agagtggaga acccatccaa cggtatcgtt aacccatccg 13440tagttagaga aagcagaatg tccacactca tgtccaagga tccagattcc gaatccgaaa 13500caagagatag agaacacgta agcagaccaa gcagcgaatc taaggaattc gttagggaga 13560agagggatgt aggtaagtcc aacgtaagcg atagcagaga tagccacgat atctctcacc 13620acgtaagaca tagacttcac gagagatctc tcgtaacagt gcttagggat agcgtcaagg 13680atatccttga tggtgtaatc tggcaccttg aaaacgtttc cgaaggtatc gatagcggtc 13740ttttgctgct tgaaagatgc aacgtttcca gaacgcctaa cggtcttagt agatccctca 13800aggatctcag atccagacac ggtaacctta gacatggtat ggtaattgta aatgtaattg 13860taatgttgtt tgttgtttgt tgttgttggt aattgttgta aaatttttgg tggtgattgg 13920ttctttaagg tgtgagagtg agttgtgagt tgtgtggtgg gtttggtgag attggggatg 13980gtgggtttat atagtggaga ctgaggaatg gggtcgtgag tgttaacttt gcatgggcta 14040cacgtgggtt cttttgggct tacacgtagt attattcatg caaatgcagc caatacatat 14100acggtatttt aataatgtgt gggaatacaa tatgccgagt attttactaa ttttggcaat 14160gacaagtgta catttggatt atcttacttg gcctctcttg ctttaatttg gattattttt 14220attctcttac cttggccgtt catattcaca tccctaaagg caagacagaa ttgaatggtg 14280gccaaaaatt aaaacgatgg atatgaccta catagtgtag gatcaattaa cgtcgaagga 14340aaatactgat tctctcaagc atacggacaa gggtaaataa catagtcacc agaacataat 14400aaacaaaaag tgcagaagca agactaaaaa aattagctat ggacattcag gttcatattg 14460gaaacatcat tatcctagtc ttgtgaccat ccttcctcct gctctagttg agaggccttg 14520ggactaacga gaggtcagtt gggatagcag atccttatcc tggactagcc tttctggtgt 14580ttcagagtct tcgtgccgcc gtctacatct atctccatta ggtctgaaga tgactcttca 14640caccaacgac gtttaaggtc tctatcctac tcctagcttg caatacctgg cttgcaatac 14700ctggagcatc gtgcacgatg attggatact gtggaggagg agtgtttgct gatttagagc 14760tcccggttgg gtgatttgac ttcgatttca gtttaggctt gttgaaattt ttcaggttcc 14820attgtgaagc ctttagagct tgagcttcct tccatgttaa tgccttgatc gaatactcct 14880agagaaaagg gaagtcgatc tctgagtatt gaaatcgaag tgcacatttt ttttcaacgt 14940gtccaatcaa tccacaaaca aagcagaaga caggtaatct ttcatactta tactgacaag

15000taatagtctt accgtcatgc ataataacgt ctcgttcctt caagaggggt tttccgacat 15060ccataacgac ccgaagcctc atgaaagcat tagggaagaa cttttggttc ttcttgtcat 15120ggcctttata ggtgtcagcc gagctcgcca attcccgtcc gactggctcc gcaaaatatt 15180cgaacggcaa gttatggact tgcaaccata actccacggt attgagcagg acctattgtg 15240aagactcatc tcatggagct tcagaatgtg gttgtcagca aaccaatgac cgaaatccat 15300cacatgacgg acgtccagtg ggtgagcgaa acgaaacagg aagcgcctat ctttcagagt 15360cgtgagctcc acaccggatt ccggcaacta cgtgttgggc aggcttcgcc gtattagaga 15420tatgttgagg cagacccatc tgtgccactc gtacaattac gagagttgtt ttttttgtga 15480ttttcctagt ttctcgttga tggtgagctc atattctaca tcgtatggtc tctcaacgtc 15540gtttcctgtc atctgatatc ccgtcatttg catccacgtg cgccgcctcc cgtgccaagt 15600ccctaggtgt catgcacgcc aaattggtgg tggtgcgggc tgccctgtgc ttcttaccga 15660tgggtggagg ttgagtttgg gggtctccgc ggcgatggta gtgggttgac ggtttggtgt 15720gggttgacgg cattgatcaa tttacttctt gcttcaaatt ctttggcaga aaacaattca 15780ttagattaga actggaaacc agagtgatga gacggattaa gtcagattcc aacagagtta 15840catctcttaa gaaataatgt aaccccttta gactttatat atttgcaatt aaaaaaataa 15900tttaactttt agactttata tatagtttta ataactaagt ttaaccactc tattatttat 15960atcgaaacta tttgtatgtc tcccctctaa ataaacttgg tattgtgttt acagaaccta 16020taatcaaata atcaatactc aactgaagtt tgtgcagtta attgaaggga ttaacggcca 16080aaatgcacta gtattatcaa ccgaatagat tcacactaga tggccatttc catcaatatc 16140atcgccgttc ttcttctgtc cacatatccc ctctgaaact tgagagacac ctgcacttca 16200ttgtccttat tacgtgttac aaaatgaaac ccatgcatcc atgcaaactg aagaatggcg 16260caagaaccct tcccctccat ttcttatgtg gcgaccatcc atttcaccat ctcccgctat 16320aaaacacccc catcacttca cctagaacat catcactact tgcttatcca tccaaaagat 16380acccactttt acaacaatta ccaacaacaa caaacaacaa acaacattac aattacattt 16440acaattacca taccatgcca cctagcgctg ctaagcaaat gggagcttct actggtgttc 16500atgctggtgt tactgactct tctgctttca ccagaaagga tgttgctgat agacctgatc 16560tcaccatcgt tggagattct gtttacgatg ctaaggcttt cagatctgag catcctggtg 16620gtgctcattt cgtttctttg ttcggaggaa gagatgctac tgaggctttc atggaatacc 16680atagaagggc ttggcctaag tctagaatgt ctagattcca cgttggatct cttgcttcta 16740ctgaggaacc tgttgctgct gatgagggat accttcaact ttgtgctagg atcgctaaga 16800tggtgccttc tgtttcttct ggattcgctc ctgcttctta ctgggttaag gctggactta 16860tccttggatc tgctatcgct cttgaggctt acatgcttta cgctggaaag agacttctcc 16920cttctatcgt tcttggatgg cttttcgctc ttatcggtct taacatccag catgatgcta 16980accatggtgc tttgtctaag tctgcttctg ttaaccttgc tcttggactt tgtcaggatt 17040ggatcggagg atctatgatc ctttggcttc aagagcatgt tgttatgcac cacctccaca 17100ctaacgatgt tgataaggat cctgatcaaa aggctcacgg tgctcttaga ctcaagccta 17160ctgatgcttg gtcacctatg cattggcttc agcatcttta ccttttgcct ggtgagacta 17220tgtacgcttt caagcttttg ttcctcgaca tctctgagct tgttatgtgg cgttgggagg 17280gtgagcctat ctctaagctt gctggatacc tctttatgcc ttctttgctt ctcaagctta 17340ccttctgggc tagattcgtt gctttgcctc tttaccttgc tccttctgtt catactgctg 17400tgtgtatcgc tgctactgtt atgactggat ctttctacct cgctttcttc ttcttcatct 17460cccacaactt cgagggtgtt gcttctgttg gacctgatgg atctatcact tctatgacta 17520gaggtgctag cttccttaag agacaagctg agacttcttc taacgttgga ggacctcttc 17580ttgctactct taacggtgga ctcaactacc aaattgagca tcacttgttc cctagagttc 17640accatggatt ctaccctaga cttgctcctc ttgttaaggc tgagcttgag gctagaggaa 17700tcgagtacaa gcactaccct actatctggt ctaaccttgc ttctaccctc agacatatgt 17760acgctcttgg aagaaggcct agatctaagg ctgagtaatg acaagcttat gtgacgtgaa 17820ataataacgg taaaatatat gtaataataa taataataaa gccacaaagt gagaatgagg 17880ggaaggggaa atgtgtaatg agccagtagc cggtggtgct aattttgtat cgtattgtca 17940ataaatcatg aattttgtgg tttttatgtg tttttttaaa tcatgaattt taaattttat 18000aaaataatct ccaatcggaa gaacaacatt ccatatccat gcatggatgt ttctttaccc 18060aaatctagtt cttgagagga tgaagcatca ccgaacagtt ctgcaactat ccctcaaaag 18120ctttaaaatg aacaacaagg aacagagcaa cgttccaaag atcccaaacg aaacatatta 18180tctatactaa tactatatta ttaattacta ctgcccggaa tcacaatccc tgaatgattc 18240ctattaacta caagccttgt tggcggcgga gaagtgatcg gcgcggcgag aagcagcgga 18300ctcggagacg aggccttgga agatctgagt cgaacgggca gaatcagtat tttccttcga 18360cgttaattga tcctacacta tgtaggtcat atccatcgtt ttaatttttg gccaccattc 18420aattctgtct tgcctttagg gatgtgaata tgaacggcca aggtaagaga ataaaaataa 18480tccaaattaa agcaagagag gccaagtaag ataatccaaa tgtacacttg tcattgccaa 18540aattagtaaa atactcggca tattgtattc ccacacatta ttaaaatacc gtatatgtat 18600tggctgcatt tgcatgaata atactacgtg taagcccaaa agaacccacg tgtagcccat 18660gcaaagttaa cactcacgac cccattcctc agtctccact atataaaccc accatcccca 18720atctcaccaa acccaccaca caactcacaa ctcactctca caccttaaag aaccaatcac 18780caccaaaaat tttacaacaa ttaccaacaa caacaaacaa caaacaacat tacaattaca 18840tttacaatta ccataccatg agcgctgtta ccgttactgg atctgatcct aagaacagag 18900gatcttctag caacaccgag caagaggttc caaaagttgc tatcgatacc aacggaaacg 18960tgttctctgt tcctgatttc accatcaagg acatccttgg agctatccct catgagtgtt 19020acgagagaag attggctacc tctctctact acgtgttcag agatatcttc tgcatgctta 19080ccaccggata ccttacccat aagatccttt accctctcct catctcttac acctctaaca 19140gcatcatcaa gttcactttc tgggcccttt acacttacgt tcaaggactt ttcggaaccg 19200gaatctgggt tctcgctcat gagtgtggac atcaagcttt ctctgattac ggaatcgtga 19260acgatttcgt tggatggacc cttcactctt accttatggt tccttacttc agctggaagt 19320actctcatgg aaagcaccat aaggctactg gacacatgac cagagatatg gttttcgttc 19380ctgccaccaa agaggaattc aagaagtcta ggaacttctt cggtaacctc gctgagtact 19440ctgaggattc tccacttaga accctttacg agcttcttgt tcaacaactt ggaggatgga 19500tcgcttacct cttcgttaac gttacaggac aaccttaccc tgatgttcct tcttggaaat 19560ggaaccactt ctggcttacc tctccacttt tcgagcaaag agatgctctc tacatcttcc 19620tttctgatct tggaatcctc acccagggaa tcgttcttac tctttggtac aagaaattcg 19680gaggatggtc ccttttcatc aactggttcg ttccttacat ctgggttaac cactggctcg 19740ttttcatcac attccttcag cacactgatc ctactatgcc tcattacaac gctgaggaat 19800ggactttcgc taagggtgct gctgctacta tcgatagaaa gttcggattc atcggacctc 19860acatcttcca tgatatcatc gagactcatg tgcttcacca ctactgttct aggatcccat 19920tctacaacgc tagacctgct tctgaggcta tcaagaaagt tatgggaaag cactacaggt 19980ctagcgacga gaacatgtgg aagtcacttt ggaagtcttt caggtcttgc caatacgttg 20040acggtgataa cggtgttctc atgttccgta acatcaacaa ctgcggagtt ggagctgctg 20100agaagtaatg aaggggtgat cgattatgag atcgtacaaa gacactgcta ggtgttaagg 20160atggataata ataataataa tgagatgaat gtgttttaag ttagtgtaac agctgtaata 20220aagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gaggctgatg 20280aaatgttatg tatgtttctt ggtttttaaa ataaatgaaa gcacatgctc gtgtggttct 20340atcgaattat tcggcggttc ctgtgggaaa aagtccagaa gggccgccgc agctactact 20400acaaccaagg ccgtggagga gggcaacaga gccagcactt cgatagctgc tgcgatgatc 20460ttaagcaatt gaggagcgag tgcacatgca ggggactgga gcgtgcaatc ggccagatga 20520ggcaggacat ccagcagcag ggacagcagc aggaagttga gaggtggtcc catcaatcta 20580aacaagtcgc tagggacctt ccgggacagt gcggcaccca gcctagccga tgccagctcc 20640aggggcagca gcagtctgca tggttttgaa gtggtgatcg atgagatcgt ataaagacac 20700tgctaggtgt taaggatggg ataataagat gtgttttaag tcattaaccg taataaaaag 20760agagagaggc tgatggaatg ttatgtatgt atgtttcttg gtttttaaaa ttaaatggaa 20820agcacatgct cgtgtgggtt ctatctcgat taaaaatccc aattatattt ggtctaattt 20880agtttggtat tgagtaaaac aaattcgaac caaaccaaaa tataaatata tagtttttat 20940atatatgcct ttaagacttt ttatagaatt ttctttaaaa aatatctaga aatatttgcg 21000actcttctgg catgtaatat ttcgttaaat atgaagtgct ccatttttat taactttaaa 21060taattggttg tacgatcact ttcttatcaa gtgttactaa aatgcgtcaa tctctttgtt 21120cttccatatt catatgtcaa aatctatcaa aattcttata tatctttttc gaatttgaag 21180tgaaatttcg ataatttaaa attaaataga acatatcatt atttaggtat catattgatt 21240tttatactta attactaaat ttggttaact ttgaaagtgt acatcaacga aaaattagtc 21300aaacgactaa aataaataaa tatcatgtgt tattaagaaa attctcctat aagaatattt 21360taatagatca tatgtttgta aaaaaaatta atttttacta acacatatat ttacttatca 21420aaaatttgac aaagtaagat taaaataata ttcatctaac aaaaaaaaaa ccagaaaatg 21480ctgaaaaccc ggcaaaaccg aaccaatcca aaccgatata gttggtttgg tttgattttg 21540atataaaccg aaccaactcg gtccatttgc acccctaatc ataatagctt taatatttca 21600agatattatt aagttaacgt tgtcaatatc ctggaaattt tgcaaaatga atcaagccta 21660tatggctgta atatgaattt aaaagcagct cgatgtggtg gtaatatgta atttacttga 21720ttctaaaaaa atatcccaag tattaataat ttctgctagg aagaaggtta gctacgattt 21780acagcaaagc cagaatacaa agaaccataa agtgattgaa gctcgaaata tacgaaggaa 21840caaatatttt taaaaaaata cgcaatgact tggaacaaaa gaaagtgata tattttttgt 21900tcttaaacaa gcatcccctc taaagaatgg cagttttcct ttgcatgtaa ctattatgct 21960cccttcgtta caaaaatttt ggactactat tgggaacttc ttctgaaaat agtcctgcag 22020gctagtagat tggttggttg gtttccatgt accagaaggc ttaccctatt agttgaaagt 22080tgaaactttg ttccctactc aattcctagt tgtgtaaatg tatgtatatg taatgtgtat 22140aaaacgtagt acttaaatga ctaggagtgg ttcttgagac cgatgagaga tgggagcaga 22200actaaagatg atgacataat taagaacgaa tttgaaaggc tcttaggttt gaatcctatt 22260cgagaatgtt tttgtcaaag atagtggcga ttttgaacca aagaaaacat ttaaaaaatc 22320agtatccggt tacgttcatg caaatagaaa gtggtctagg atctgattgt aattttagac 22380ttaaagagtc tcttaagatt caatcctggc tgtgtacaaa actacaaata atatatttta 22440gactatttgg ccttaactaa acttccactc attatttact gaggttagag aatagacttg 22500cgaataaaca cattcccgag aaatactcat gatcccataa ttagtcagag ggtatgccaa 22560tcagatctaa gaacacacat tccctcaaat tttaatgcac atgtaatcat agtttagcac 22620aattcaaaaa taatgtagta ttaaagacag aaatttgtag actttttttt ggcgttaaaa 22680gaagactaag tttatacgta cattttattt taagtggaaa accgaaattt tccatcgaaa 22740tatatgaatt tagtatatat atttctgcaa tgtactattt tgctattttg gcaactttca 22800gtggactact actttattac aatgtgtatg gatgcatgag tttgagtata cacatgtcta 22860aatgcatgct ttgtaaaacg taacggacca caaaagagga tccatacaaa tacatctcat 22920agcttcctcc attattttcc gacacaaaca gagcatttta caacaattac caacaacaac 22980aaacaacaaa caacattaca attacattta caattaccat accatggaat ttgctcaacc 23040tctcgttgct atggctcaag agcagtacgc tgctatcgat gctgttgttg ctcctgctat 23100cttctctgct accgactcta ttggatgggg actcaagcct atctcttctg ctactaagga 23160tctccctctc gttgaatctc ctacccctct tatcctttct ctcctcgctt acttcgctat 23220cgttggttct ggactcgttt accgtaaagt gttccctaga accgttaagg gacaggatcc 23280tttccttctc aaggctctta tgctcgctca caacgttttc cttatcggac tcagccttta 23340catgtgcctc aagctcgttt acgaggctta cgtgaacaag tactccttct ggggaaacgc 23400ttacaaccct gctcaaaccg agatggctaa ggtgatctgg atcttctacg tgtccaagat 23460ctacgagttc atggacacct tcatcatgct tctcaaggga aacgttaacc aggtttcctt 23520cctccatgtt taccaccacg gatctatctc tggaatctgg tggatgatca cttatgctgc 23580tccaggtgga gatgcttact tctctgctgc tctcaactct tgggttcatg tgtgcatgta 23640cacctactac ttcatggctg ctgttcttcc taaggacgaa aagaccaaga gaaagtacct 23700ttggtgggga agatacctta cccagatgca aatgttccag ttcttcatga accttctcca 23760ggctgtttac ctcctctact cttcttctcc ttaccctaag ttcattgctc aactcctcgt 23820tgtttacatg gttaccctcc tcatgctttt cggaaacttc tactacatga agcaccacgc 23880ttctaagtga taagggccgc cgccatgtga cagatcgaag gaagaaagtg taataagacg 23940actctcacta ctcgatcgct agtgattgtc attgttatat ataataatgt tatctttcac 24000aacttatcgt aatgcatgtg aaactataac acattaatcc tacttgtcat atgataacac 24060tctccccatt taaaactctt gtcaatttaa agatataaga ttctttaaat gattaaaaaa 24120aatatattat aaattcaatc actcctacta ataaattatt aattattatt tattgattaa 24180aaaaatactt atactaattt agtctgaata gaataattag attctagcct gcagggcggc 24240cgcggatccc atggagtcaa agattcaaat agaggaccta acagaactcg ccgtaaagac 24300tggcgaacag ttcatacaga gtctcttacg actcaatgac aagaagaaaa tcttcgtcaa 24360catggtggag cacgacacac ttgtctactc caaaaatatc aaagatacag tctcagaaga 24420ccaaagggca attgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca 24480ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa 24540atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg acagtggtcc 24600caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc 24660ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca 24720ctatccttcg caagaccctt cctctatata aggaagttca tttcatttgg agagaacacg 24780ggggactgaa ttaaatatga gccctgagag gcgtcctgtt gaaatcagac ctgctactgc 24840tgctgatatg gctgctgttt gtgatatcgt gaaccactac atcgagactt ctaccgttaa 24900cttcagaact gagcctcaaa ctcctcaaga gtggatcgat gatcttgaga gactccaaga 24960tagataccct tggcttgttg ctgaggttga gggtgttgtt gctggaatcg cttacgctgg 25020accttggaag gctagaaacg cttacgattg gactgttgag tctaccgttt acgtttcaca 25080cagacatcag agacttggac ttggatctac cctttacact caccttctca agtctatgga 25140agctcaggga ttcaagtctg ttgttgctgt tatcggactc cctaacgatc cttctgttag 25200acttcatgag gctcttggat acactgctag aggaactctt agagctgctg gatacaagca 25260cggtggatgg catgatgttg gattctggca aagagatttc gagcttcctg ctcctcctag 25320acctgttaga ccagttactc agatctgaat ttgcgtgatc gttcaaacat ttggcaataa 25380agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 25440aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 25500tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 25560gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatca ctagtgatgt 25620acggttaaaa ccaccccagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag 25680cgtcaatttg tttacaccac aatatatcct gccaccagcc agccaacagc tccccgaccg 25740gcagctcggc acaaaatcac cactcgatac aggcagccca tcagtcc 25787422824DNAArtificial SequencepGA7- mod_D nucleotide sequence 4tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180tcacgacgtt gtaaaacggg cgcctcgatt aaaaatccca attatatttg gtctaattta 240gtttggtatt gagtaaaaca aattcgaacc aaaccaaaat ataaatatat agtttttata 300tatatgcctt taagactttt tatagaattt tctttaaaaa atatctagaa atatttgcga 360ctcttctggc atgtaatatt tcgttaaata tgaagtgctc catttttatt aactttaaat 420aattggttgt acgatcactt tcttatcaag tgttactaaa atgcgtcaat ctctttgttc 480ttccatattc atatgtcaaa atctatcaaa attcttatat atctttttcg aatttgaagt 540gaaatttcga taatttaaaa ttaaatagaa catatcatta tttaggtatc atattgattt 600ttatacttaa ttactaaatt tggttaactt tgaaagtgta catcaacgaa aaattagtca 660aacgactaaa ataaataaat atcatgtgtt attaagaaaa ttctcctata agaatatttt 720aatagatcat atgtttgtaa aaaaaattaa tttttactaa cacatatatt tacttatcaa 780aaatttgaca aagtaagatt aaaataatat tcatctaaca aaaaaaaaac cagaaaatgc 840tgaaaacccg gcaaaaccga accaatccaa accgatatag ttggtttggt ttgattttga 900tataaaccga accaactcgg tccatttgca cccctaatca taatagcttt aatatttcaa 960gatattatta agttaacgtt gtcaatatcc tggaaatttt gcaaaatgaa tcaagcctat 1020atggctgtaa tatgaattta aaagcagctc gatgtggtgg taatatgtaa tttacttgat 1080tctaaaaaaa tatcccaagt attaataatt tctgctagga agaaggttag ctacgattta 1140cagcaaagcc agaatacaaa gaaccataaa gtgattgaag ctcgaaatat acgaaggaac 1200aaatattttt aaaaaaatac gcaatgactt ggaacaaaag aaagtgatat attttttgtt 1260cttaaacaag catcccctct aaagaatggc agttttcctt tgcatgtaac tattatgctc 1320ccttcgttac aaaaattttg gactactatt gggaacttct tctgaaaata gtggcgcccc 1380gcggaaagct tgctagccaa ttggggccca acgttctcga gtttttctag aaggaaactg 1440aaggcgggaa acgacaatct gctagtggat ctcccagtca cgacgttgta aaacgggcgc 1500cccgcggaaa gcttgcggcc gcccgatcta gtaacataga tgacaccgcg cgcgataatt 1560tatcctagtt tgcgcgctat attttgtttt ctatcgcgta ttaaatgtat aattgcggga 1620ctctaatcat aaaaacccat ctcataaata acgtcatgca ttacatgtta attattacgt 1680gcttaacgta attcaacaga aattatatga taatcatcgc aagaccggca acaggattca 1740atcttaagaa actttattgc caaatgtttg aacgatcggc gcgcctcatt agtgagcctt 1800ctcagccttt ccgttaacgt agtagtgctg tcccacctta tcaaggttag agaaagtagc 1860cttccaagca ccgtagtaag agagcacctt gtagttgagt ccccacttct tagcgaaagg 1920aacgaatctt ctgctaacct caggctgtct gaattgaggc atatcaggga agaggtggtg 1980gataacctga cagttaaggt atcccataag ccagttcacg tatcctctag aaggatcgat 2040atcaacggtg tgatcaacag cgtagttaac ccaagaaagg tgcttatcag atggaacaac 2100agggaggtga gtatgagaag tagagaagtg agcgaaaagg tacatgtaag cgatccagtt 2160tccgaaagtg aaccaccagt aagcaacagg ccaagagtat ccagtagcaa gcttgataac 2220agcggttcta acaacatgag aaacgagcat ccaagaagcc tcttcgtagt tcttcttacg 2280gagaacttgt ctagggtgga gaacgtagat ccagaaagct tgaacaagaa gtccagaggt 2340aacaggaacg aaagtccaag cttgaagtct agcccaagct ctagagaatc ctctaggtct 2400gttatcctca acagcagtgt tgaagaaagc cacagcagga gtggtatcaa gatccatatc 2460gtgtctaacc ttttgagggg tagcatggtg cttgttatgc atctggttcc acatctcacc 2520agaagtagaa agtccgaatc cacaagtcat agcctgaagt ctcttgtcca cgtaaacaga 2580tccggtaaga gagttatgtc caccctcatg ttgaacccat ccacatctag ctccgaagaa 2640agcaccgtaa acaacagaag caatgatagg gtatccagcg tacataagag cagttccaag 2700agcgaatgta gcaagaagct cgagaagtct gtaagccaca tgggtgatag aaggcttgaa 2760gaatccatct ctctcaagct cagcacgcca tctagcgaaa tcctcaagca taggagcatc 2820ctcagactca gatctcttga tctcagcagg tctagaaggc aaagctctaa gcatcttcca 2880agccttgaga gaacgcatgt ggaattcttt gaaagcctca gtagcatcag caccagtgtt 2940agcaagcatg tagaagatca cagatccacc agggtgcttg aagttagtca catcgtactc 3000aacgtcctca actctaaccc atctagtctc gaaagtagca gcaagctcat gaggctcaag 3060agtcttaaga tcaacaggag cagtagaagc atccttagca tcaagagcct cagcagaaga 3120tttagacctg gtaagtggag atctaggaga agatcttcca tcagtcttag gagggcacat 3180ggtatggtaa ttgtaaatgt aattgtaatg ttgtttgttg tttgttgttg ttggtaattg 3240ttgtaaaaga tcctcgtgta tgtttttaat cttgtttgta tcgatgagtt ttggtttgag 3300taaagagtga agcggatgag ttaatttata ggctataaag gagatttgca tggcgatcac 3360gtgtaataat gcatgcacgc atgtgattgt atgtgtgtgc tgtgagagag aagctcttag 3420gtgtttgaag ggagtgacaa gtggcgaaga aaaacaattc tccgcggctg catgctatgt 3480gtaacgtgta gctaatgttc tggcatggca tcttatgaac gattcttttt aaaaacaagg 3540taaaaactta acttcataaa attaaaaaaa aaaacgttta ctaagttggt ttaaaagggg 3600atgagactag tagattggtt ggttggtttc catgtaccag aaggcttacc ctattagttg 3660aaagttgaaa ctttgttccc tactcaattc ctagttgtgt aaatgtatgt atatgtaatg 3720tgtataaaac gtagtactta aatgactagg agtggttctt gagaccgatg agagatggga 3780gcagaactaa agatgatgac ataattaaga acgaatttga aaggctctta ggtttgaatc 3840ctattcgaga atgtttttgt caaagatagt ggcgattttg aaccaaagaa aacatttaaa 3900aaatcagtat ccggttacgt tcatgcaaat agaaagtggt ctaggatctg attgtaattt 3960tagacttaaa gagtctctta agattcaatc ctggctgtgt acaaaactac aaataatata 4020ttttagacta tttggcctta actaaacttc cactcattat ttactgaggt tagagaatag 4080acttgcgaat aaacacattc ccgagaaata ctcatgatcc cataattagt cagagggtat 4140gccaatcaga tctaagaaca cacattccct caaattttaa tgcacatgta atcatagttt

4200agcacaattc aaaaataatg tagtattaaa gacagaaatt tgtagacttt tttttggcgt 4260taaaagaaga ctaagtttat acgtacattt tattttaagt ggaaaaccga aattttccat 4320cgaaatatat gaatttagta tatatatttc tgcaatgtac tattttgcta ttttggcaac 4380tttcagtgga ctactacttt attacaatgt gtatggatgc atgagtttga gtatacacat 4440gtctaaatgc atgctttgta aaacgtaacg gaccacaaaa gaggatccat acaaatacat 4500ctcatagctt cctccattat tttccgacac aaacagagca ttttacaaca attaccaaca 4560acaacaaaca acaaacaaca ttacaattac atttacaatt accataccat ggaattcgcc 4620cagcctcttg ttgctatggc tcaagagcaa tacgctgcta tcgatgctgt tgttgctcct 4680gctatcttct ctgctactga ttctatcgga tggggactta agcctatctc ttctgctact 4740aaggacttgc ctcttgttga gtctcctaca cctctcatcc tttctttgct tgcttacttc 4800gctatcgttg gatctggact cgtttacaga aaggttttcc ctagaaccgt gaagggacaa 4860gatccattcc ttttgaaggc tcttatgctt gctcacaacg tgttccttat cggactttct 4920ctttacatgt gcctcaagct tgtgtacgag gcttacgtta acaagtactc tttctgggga 4980aacgcttaca accctgctca aactgagatg gctaaggtta tctggatctt ctacgtgagc 5040aagatctacg agttcatgga taccttcatc atgctcctca agggaaatgt taaccaggtt 5100agcttccttc acgtttacca tcacggatct atctctggaa tctggtggat gattacttac 5160gctgctcctg gtggtgatgc ttacttctct gctgctctta actcttgggt tcacgtgtgt 5220atgtacacct actattttat ggctgccgtg cttcctaagg acgagaaaac taagagaaag 5280tacctctggt ggggaagata ccttactcaa atgcagatgt tccagttctt catgaacctt 5340ctccaggctg tttaccttct ctactcttca tctccttacc ctaagtttat cgctcagctc 5400ctcgtggtgt acatggttac tcttctcatg cttttcggaa acttctacta catgaagcac 5460cacgctagca agtgatgagg cgcgccgggc cgccgccatg tgacagatcg aaggaagaaa 5520gtgtaataag acgactctca ctactcgatc gctagtgatt gtcattgtta tatataataa 5580tgttatcttt cacaacttat cgtaatgcat gtgaaactat aacacattaa tcctacttgt 5640catatgataa cactctcccc atttaaaact cttgtcaatt taaagatata agattcttta 5700aatgattaaa aaaaatatat tataaattca atcactccta ctaataaatt attaattatt 5760atttattgat taaaaaaata cttatactaa tttagtctga atagaataat tagattctag 5820tctcatcccc ttttaaacca acttagtaaa cgtttttttt tttaatttta tgaagttaag 5880tttttacctt gtttttaaaa agaatcgttc ataagatgcc atgccagaac attagctaca 5940cgttacacat agcatgcagc cgcggagaat tgtttttctt cgccacttgt cactcccttc 6000aaacacctaa gagcttctct ctcacagcac acacatacaa tcacatgcgt gcatgcatta 6060ttacacgtga tcgccatgca aatctccttt atagcctata aattaactca tccgcttcac 6120tctttactca aaccaaaact catcgataca aacaagatta aaaacataca cgaggatctt 6180ttacaacaat taccaacaac aacaaacaac aaacaacatt acaattacat ttacaattac 6240cataccatgc ctccaaggga ctcttactct tatgctgctc ctccttctgc tcaacttcac 6300gaagttgata ctcctcaaga gcacgacaag aaagagcttg ttatcggaga tagggcttac 6360gatgttacca acttcgttaa gagacaccct ggtggaaaga tcattgctta ccaagttgga 6420actgatgcta ccgatgctta caagcagttc catgttagat ctgctaaggc tgacaagatg 6480cttaagtctc ttccttctcg tcctgttcac aagggatact ctccaagaag ggctgatctt 6540atcgctgatt tccaagagtt caccaagcaa cttgaggctg agggaatgtt cgagccttct 6600cttcctcatg ttgcttacag acttgctgag gttatcgcta tgcatgttgc tggtgctgct 6660cttatctggc atggatacac tttcgctgga atcgctatgc ttggagttgt tcagggaaga 6720tgtggatggc ttatgcatga gggtggacat tactctctca ctggaaacat tgctttcgac 6780agagctatcc aagttgcttg ttacggactt ggatgtggaa tgtctggtgc ttggtggcgt 6840aaccagcata acaagcacca tgctactcct caaaagcttc agcacgatgt tgatcttgat 6900acccttcctc tcgttgcttt ccatgagaga atcgctgcta aggttaagtc tcctgctatg 6960aaggcttggc tttctatgca agctaagctt ttcgctcctg ttaccactct tcttgttgct 7020cttggatggc agctttacct tcatcctaga cacatgctca ggactaagca ctacgatgag 7080cttgctatgc tcggaatcag atacggactt gttggatacc ttgctgctaa ctacggtgct 7140ggatacgttc tcgcttgtta ccttctttac gttcagcttg gagctatgta catcttctgc 7200aacttcgctg tttctcatac tcacctccct gttgttgagc ctaacgagca tgctacttgg 7260gttgagtacg ctgctaacca cactactaac tgttctccat cttggtggtg tgattggtgg 7320atgtcttacc ttaactacca gatcgagcac cacctttacc cttctatgcc tcaattcaga 7380caccctaaga tcgctcctag agttaagcag cttttcgaga agcacggact tcactacgat 7440gttagaggat acttcgaggc tatggctgat actttcgcta accttgataa cgttgcccat 7500gctcctgaga agaaaatgca gtaatgagat cgttcaaaca tttggcaata aagtttctta 7560agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 7620aagcacgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 7680agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 7740gataaattat cgcgcgcggt gtcatctatg ttactagatc ggtcgattaa aaatcccaat 7800tatatttggt ctaatttagt ttggtattga gtaaaacaaa ttcgaaccaa accaaaatat 7860aaatatatag tttttatata tatgccttta agacttttta tagaattttc tttaaaaaat 7920atctagaaat atttgcgact cttctggcat gtaatatttc gttaaatatg aagtgctcca 7980tttttattaa ctttaaataa ttggttgtac gatcactttc ttatcaagtg ttactaaaat 8040gcgtcaatct ctttgttctt ccatattcat atgtcaaaat ctatcaaaat tcttatatat 8100ctttttcgaa tttgaagtga aatttcgata atttaaaatt aaatagaaca tatcattatt 8160taggtatcat attgattttt atacttaatt actaaatttg gttaactttg aaagtgtaca 8220tcaacgaaaa attagtcaaa cgactaaaat aaataaatat catgtgttat taagaaaatt 8280ctcctataag aatattttaa tagatcatat gtttgtaaaa aaaattaatt tttactaaca 8340catatattta cttatcaaaa atttgacaaa gtaagattaa aataatattc atctaacaaa 8400aaaaaaacca gaaaatgctg aaaacccggc aaaaccgaac caatccaaac cgatatagtt 8460ggtttggttt gattttgata taaaccgaac caactcggtc catttgcacc cctaatcata 8520atagctttaa tatttcaaga tattattaag ttaacgttgt caatatcctg gaaattttgc 8580aaaatgaatc aagcctatat ggctgtaata tgaatttaaa agcagctcga tgtggtggta 8640atatgtaatt tacttgattc taaaaaaata tcccaagtat taataatttc tgctaggaag 8700aaggttagct acgatttaca gcaaagccag aatacaaaga accataaagt gattgaagct 8760cgaaatatac gaaggaacaa atatttttaa aaaaatacgc aatgacttgg aacaaaagaa 8820agtgatatat tttttgttct taaacaagca tcccctctaa agaatggcag ttttcctttg 8880catgtaacta ttatgctccc ttcgttacaa aaattttgga ctactattgg gaacttcttc 8940tgaaaatagt gatagaaccc acacgagcat gtgctttcca tttaatttta aaaaccaaga 9000aacatacata cataacattc catcagcctc tctctctttt tattacggtt aatgacttaa 9060aacacatctt attatcccat ccttaacacc tagcagtgtc tttatacgat ctcatcgatc 9120accacttcaa aaccatgcag actgctgctg cccctggagc tggcatcggc taggctgggt 9180gccgcactgt cccggaaggt ccctagcgac ttgtttagat tgatgggacc acctctcaac 9240ttcctgctgc tgtccctgct gctggatgtc ctgcctcatc tggccgattg cacgctccag 9300tcccctgcat gtgcactcgc tcctcaattg cttaagatca tcgcagcagc tatcgaagtg 9360ctggctctgt tgccctcctc cacggccttg gttgtagtag tagctgccgc cgcccttctg 9420gactttttcc cacaggaacc gccgaataat tcgatagaac cacacgagca tgtgctttca 9480tttattttaa aaaccaagaa acatacataa catttcatca gcctctctct ctctctctct 9540ctctctctct ctctctctct ctctctctct ctctttatta cagctgttac actaacttaa 9600aacacattca tctcattatt attattatta tccatcctta acacctagca gtgtctttgt 9660acgatctcat aatcgatcac cccttcatca ggtatcctta ggcttcactc caacgttgtt 9720gcagttacgg aacatgtaca caccatcatg gttctcaacg aactggcaag atctccaagt 9780tttccaaagg ctaacccaca tgttctcatc ggtgtgtctg tagtgctctc ccataacttt 9840cttgatgcac tcggtagctt ctctagcatg gtagaatggg atccttgaaa cgtagtgatg 9900gagcacatga gtctcgatga tgtcatggaa gatgattccg aggattccga actctctatc 9960gatagtagca gcagcaccct tagcgaaagt ccactcttga gcatcgtaat gaggcataga 10020agaatcggtg tgctgaagga aggtaacgaa aacaagccag tggttaacaa ggatccaagg 10080acagaaccat gtgatgaaag taggccagaa tccgaaaacc ttgtaagcgg tgtaaacaga 10140agtgagggta gcaaggattc caagatcaga aagaacgatg taccagtagt ccttcttatc 10200gaaaacaggg ctagaaggcc agtagtgaga cttgaagaac ttagaaacac cagggtaagg 10260ttgtccagta gcgttagtag caaggtaaag agaaagtcct ccaagctgtt ggaacaagag 10320agcgaaaaca gagtagatag gagtttcctc agcgatatcg tgaaggctgg taacttggtg 10380cttctctttg aattcctcgg cggtgtaagg aacgaaaacc atatctctgg tcatgtgtcc 10440agtagcctta tggtgcttag catgagagaa cttccagctg aagtaaggaa ccataacaag 10500agagtggaga acccatccaa cggtatcgtt aacccatccg tagttagaga aagcagaatg 10560tccacactca tgtccaagga tccagattcc gaatccgaaa caagagatag agaacacgta 10620agcagaccaa gcagcgaatc taaggaattc gttagggaga agagggatgt aggtaagtcc 10680aacgtaagcg atagcagaga tagccacgat atctctcacc acgtaagaca tagacttcac 10740gagagatctc tcgtaacagt gcttagggat agcgtcaagg atatccttga tggtgtaatc 10800tggcaccttg aaaacgtttc cgaaggtatc gatagcggtc ttttgctgct tgaaagatgc 10860aacgtttcca gaacgcctaa cggtcttagt agatccctca aggatctcag atccagacac 10920ggtaacctta gacatggtat ggtaattgta aatgtaattg taatgttgtt tgttgtttgt 10980tgttgttggt aattgttgta aaatttttgg tggtgattgg ttctttaagg tgtgagagtg 11040agttgtgagt tgtgtggtgg gtttggtgag attggggatg gtgggtttat atagtggaga 11100ctgaggaatg gggtcgtgag tgttaacttt gcatgggcta cacgtgggtt cttttgggct 11160tacacgtagt attattcatg caaatgcagc caatacatat acggtatttt aataatgtgt 11220gggaatacaa tatgccgagt attttactaa ttttggcaat gacaagtgta catttggatt 11280atcttacttg gcctctcttg ctttaatttg gattattttt attctcttac cttggccgtt 11340catattcaca tccctaaagg caagacagaa ttgaatggtg gccaaaaatt aaaacgatgg 11400atatgaccta catagtgtag gatcaattaa cgtcgaagga aaatactgat tctctcaagc 11460atacggacaa gggtaaataa catagtcacc agaacataat aaacaaaaag tgcagaagca 11520agactaaaaa aattagctat ggacattcag gttcatattg gaaacatcat tatcctagtc 11580ttgtgaccat ccttcctcct gctctagttg agaggccttg ggactaacga gaggtcagtt 11640gggatagcag atccttatcc tggactagcc tttctggtgt ttcagagtct tcgtgccgcc 11700gtctacatct atctccatta ggtctgaaga tgactcttca caccaacgac gtttaaggtc 11760tctatcctac tcctagcttg caatacctgg cttgcaatac ctggagcatc gtgcacgatg 11820attggatact gtggaggagg agtgtttgct gatttagagc tcccggttgg gtgatttgac 11880ttcgatttca gtttaggctt gttgaaattt ttcaggttcc attgtgaagc ctttagagct 11940tgagcttcct tccatgttaa tgccttgatc gaatactcct agagaaaagg gaagtcgatc 12000tctgagtatt gaaatcgaag tgcacatttt ttttcaacgt gtccaatcaa tccacaaaca 12060aagcagaaga caggtaatct ttcatactta tactgacaag taatagtctt accgtcatgc 12120ataataacgt ctcgttcctt caagaggggt tttccgacat ccataacgac ccgaagcctc 12180atgaaagcat tagggaagaa cttttggttc ttcttgtcat ggcctttata ggtgtcagcc 12240gagctcgcca attcccgtcc gactggctcc gcaaaatatt cgaacggcaa gttatggact 12300tgcaaccata actccacggt attgagcagg acctattgtg aagactcatc tcatggagct 12360tcagaatgtg gttgtcagca aaccaatgac cgaaatccat cacatgacgg acgtccagtg 12420ggtgagcgaa acgaaacagg aagcgcctat ctttcagagt cgtgagctcc acaccggatt 12480ccggcaacta cgtgttgggc aggcttcgcc gtattagaga tatgttgagg cagacccatc 12540tgtgccactc gtacaattac gagagttgtt ttttttgtga ttttcctagt ttctcgttga 12600tggtgagctc atattctaca tcgtatggtc tctcaacgtc gtttcctgtc atctgatatc 12660ccgtcatttg catccacgtg cgccgcctcc cgtgccaagt ccctaggtgt catgcacgcc 12720aaattggtgg tggtgcgggc tgccctgtgc ttcttaccga tgggtggagg ttgagtttgg 12780gggtctccgc ggcgatggta gtgggttgac ggtttggtgt gggttgacgg cattgatcaa 12840tttacttctt gcttcaaatt ctttggcaga aaacaattca ttagattaga actggaaacc 12900agagtgatga gacggattaa gtcagattcc aacagagtta catctcttaa gaaataatgt 12960aaccccttta gactttatat atttgcaatt aaaaaaataa tttaactttt agactttata 13020tatagtttta ataactaagt ttaaccactc tattatttat atcgaaacta tttgtatgtc 13080tcccctctaa ataaacttgg tattgtgttt acagaaccta taatcaaata atcaatactc 13140aactgaagtt tgtgcagtta attgaaggga ttaacggcca aaatgcacta gtattatcaa 13200ccgaatagat tcacactaga tggccatttc catcaatatc atcgccgttc ttcttctgtc 13260cacatatccc ctctgaaact tgagagacac ctgcacttca ttgtccttat tacgtgttac 13320aaaatgaaac ccatgcatcc atgcaaactg aagaatggcg caagaaccct tcccctccat 13380ttcttatgtg gcgaccatcc atttcaccat ctcccgctat aaaacacccc catcacttca 13440cctagaacat catcactact tgcttatcca tccaaaagat acccactttt acaacaatta 13500ccaacaacaa caaacaacaa acaacattac aattacattt acaattacca taccatgcca 13560cctagcgctg ctaagcaaat gggagcttct actggtgttc atgctggtgt tactgactct 13620tctgctttca ccagaaagga tgttgctgat agacctgatc tcaccatcgt tggagattct 13680gtttacgatg ctaaggcttt cagatctgag catcctggtg gtgctcattt cgtttctttg 13740ttcggaggaa gagatgctac tgaggctttc atggaatacc atagaagggc ttggcctaag 13800tctagaatgt ctagattcca cgttggatct cttgcttcta ctgaggaacc tgttgctgct 13860gatgagggat accttcaact ttgtgctagg atcgctaaga tggtgccttc tgtttcttct 13920ggattcgctc ctgcttctta ctgggttaag gctggactta tccttggatc tgctatcgct 13980cttgaggctt acatgcttta cgctggaaag agacttctcc cttctatcgt tcttggatgg 14040cttttcgctc ttatcggtct taacatccag catgatgcta accatggtgc tttgtctaag 14100tctgcttctg ttaaccttgc tcttggactt tgtcaggatt ggatcggagg atctatgatc 14160ctttggcttc aagagcatgt tgttatgcac cacctccaca ctaacgatgt tgataaggat 14220cctgatcaaa aggctcacgg tgctcttaga ctcaagccta ctgatgcttg gtcacctatg 14280cattggcttc agcatcttta ccttttgcct ggtgagacta tgtacgcttt caagcttttg 14340ttcctcgaca tctctgagct tgttatgtgg cgttgggagg gtgagcctat ctctaagctt 14400gctggatacc tctttatgcc ttctttgctt ctcaagctta ccttctgggc tagattcgtt 14460gctttgcctc tttaccttgc tccttctgtt catactgctg tgtgtatcgc tgctactgtt 14520atgactggat ctttctacct cgctttcttc ttcttcatct cccacaactt cgagggtgtt 14580gcttctgttg gacctgatgg atctatcact tctatgacta gaggtgctag cttccttaag 14640agacaagctg agacttcttc taacgttgga ggacctcttc ttgctactct taacggtgga 14700ctcaactacc aaattgagca tcacttgttc cctagagttc accatggatt ctaccctaga 14760cttgctcctc ttgttaaggc tgagcttgag gctagaggaa tcgagtacaa gcactaccct 14820actatctggt ctaaccttgc ttctaccctc agacatatgt acgctcttgg aagaaggcct 14880agatctaagg ctgagtaatg acaagcttat gtgacgtgaa ataataacgg taaaatatat 14940gtaataataa taataataaa gccacaaagt gagaatgagg ggaaggggaa atgtgtaatg 15000agccagtagc cggtggtgct aattttgtat cgtattgtca ataaatcatg aattttgtgg 15060tttttatgtg tttttttaaa tcatgaattt taaattttat aaaataatct ccaatcggaa 15120gaacaacatt ccatatccat gcatggatgt ttctttaccc aaatctagtt cttgagagga 15180tgaagcatca ccgaacagtt ctgcaactat ccctcaaaag ctttaaaatg aacaacaagg 15240aacagagcaa cgttccaaag atcccaaacg aaacatatta tctatactaa tactatatta 15300ttaattacta ctgcccggaa tcacaatccc tgaatgattc ctattaacta caagccttgt 15360tggcggcgga gaagtgatcg gcgcggcgag aagcagcgga ctcggagacg aggccttgga 15420agatctgagt cgaacgggca gaatcagtat tttccttcga cgttaattga tcctacacta 15480tgtaggtcat atccatcgtt ttaatttttg gccaccattc aattctgtct tgcctttagg 15540gatgtgaata tgaacggcca aggtaagaga ataaaaataa tccaaattaa agcaagagag 15600gccaagtaag ataatccaaa tgtacacttg tcattgccaa aattagtaaa atactcggca 15660tattgtattc ccacacatta ttaaaatacc gtatatgtat tggctgcatt tgcatgaata 15720atactacgtg taagcccaaa agaacccacg tgtagcccat gcaaagttaa cactcacgac 15780cccattcctc agtctccact atataaaccc accatcccca atctcaccaa acccaccaca 15840caactcacaa ctcactctca caccttaaag aaccaatcac caccaaaaat tttacaacaa 15900ttaccaacaa caacaaacaa caaacaacat tacaattaca tttacaatta ccataccatg 15960agcgctgtta ccgttactgg atctgatcct aagaacagag gatcttctag caacaccgag 16020caagaggttc caaaagttgc tatcgatacc aacggaaacg tgttctctgt tcctgatttc 16080accatcaagg acatccttgg agctatccct catgagtgtt acgagagaag attggctacc 16140tctctctact acgtgttcag agatatcttc tgcatgctta ccaccggata ccttacccat 16200aagatccttt accctctcct catctcttac acctctaaca gcatcatcaa gttcactttc 16260tgggcccttt acacttacgt tcaaggactt ttcggaaccg gaatctgggt tctcgctcat 16320gagtgtggac atcaagcttt ctctgattac ggaatcgtga acgatttcgt tggatggacc 16380cttcactctt accttatggt tccttacttc agctggaagt actctcatgg aaagcaccat 16440aaggctactg gacacatgac cagagatatg gttttcgttc ctgccaccaa agaggaattc 16500aagaagtcta ggaacttctt cggtaacctc gctgagtact ctgaggattc tccacttaga 16560accctttacg agcttcttgt tcaacaactt ggaggatgga tcgcttacct cttcgttaac 16620gttacaggac aaccttaccc tgatgttcct tcttggaaat ggaaccactt ctggcttacc 16680tctccacttt tcgagcaaag agatgctctc tacatcttcc tttctgatct tggaatcctc 16740acccagggaa tcgttcttac tctttggtac aagaaattcg gaggatggtc ccttttcatc 16800aactggttcg ttccttacat ctgggttaac cactggctcg ttttcatcac attccttcag 16860cacactgatc ctactatgcc tcattacaac gctgaggaat ggactttcgc taagggtgct 16920gctgctacta tcgatagaaa gttcggattc atcggacctc acatcttcca tgatatcatc 16980gagactcatg tgcttcacca ctactgttct aggatcccat tctacaacgc tagacctgct 17040tctgaggcta tcaagaaagt tatgggaaag cactacaggt ctagcgacga gaacatgtgg 17100aagtcacttt ggaagtcttt caggtcttgc caatacgttg acggtgataa cggtgttctc 17160atgttccgta acatcaacaa ctgcggagtt ggagctgctg agaagtaatg aaggggtgat 17220cgattatgag atcgtacaaa gacactgcta ggtgttaagg atggataata ataataataa 17280tgagatgaat gtgttttaag ttagtgtaac agctgtaata aagagagaga gagagagaga 17340gagagagaga gagagagaga gagagagaga gaggctgatg aaatgttatg tatgtttctt 17400ggtttttaaa ataaatgaaa gcacatgctc gtgtggttct atcgaattat tcggcggttc 17460ctgtgggaaa aagtccagaa gggccgccgc agctactact acaaccaagg ccgtggagga 17520gggcaacaga gccagcactt cgatagctgc tgcgatgatc ttaagcaatt gaggagcgag 17580tgcacatgca ggggactgga gcgtgcaatc ggccagatga ggcaggacat ccagcagcag 17640ggacagcagc aggaagttga gaggtggtcc catcaatcta aacaagtcgc tagggacctt 17700ccgggacagt gcggcaccca gcctagccga tgccagctcc aggggcagca gcagtctgca 17760tggttttgaa gtggtgatcg atgagatcgt ataaagacac tgctaggtgt taaggatggg 17820ataataagat gtgttttaag tcattaaccg taataaaaag agagagaggc tgatggaatg 17880ttatgtatgt atgtttcttg gtttttaaaa ttaaatggaa agcacatgct cgtgtgggtt 17940ctatctcgat taaaaatccc aattatattt ggtctaattt agtttggtat tgagtaaaac 18000aaattcgaac caaaccaaaa tataaatata tagtttttat atatatgcct ttaagacttt 18060ttatagaatt ttctttaaaa aatatctaga aatatttgcg actcttctgg catgtaatat 18120ttcgttaaat atgaagtgct ccatttttat taactttaaa taattggttg tacgatcact 18180ttcttatcaa gtgttactaa aatgcgtcaa tctctttgtt cttccatatt catatgtcaa 18240aatctatcaa aattcttata tatctttttc gaatttgaag tgaaatttcg ataatttaaa 18300attaaataga acatatcatt atttaggtat catattgatt tttatactta attactaaat 18360ttggttaact ttgaaagtgt acatcaacga aaaattagtc aaacgactaa aataaataaa 18420tatcatgtgt tattaagaaa attctcctat aagaatattt taatagatca tatgtttgta 18480aaaaaaatta atttttacta acacatatat ttacttatca aaaatttgac aaagtaagat 18540taaaataata ttcatctaac aaaaaaaaaa ccagaaaatg ctgaaaaccc ggcaaaaccg 18600aaccaatcca aaccgatata gttggtttgg tttgattttg atataaaccg aaccaactcg 18660gtccatttgc acccctaatc ataatagctt taatatttca agatattatt aagttaacgt 18720tgtcaatatc ctggaaattt tgcaaaatga atcaagccta tatggctgta atatgaattt 18780aaaagcagct cgatgtggtg gtaatatgta atttacttga ttctaaaaaa atatcccaag 18840tattaataat ttctgctagg aagaaggtta gctacgattt acagcaaagc cagaatacaa 18900agaaccataa agtgattgaa gctcgaaata tacgaaggaa caaatatttt taaaaaaata 18960cgcaatgact tggaacaaaa gaaagtgata tattttttgt tcttaaacaa gcatcccctc 19020taaagaatgg cagttttcct ttgcatgtaa ctattatgct cccttcgtta caaaaatttt 19080ggactactat tgggaacttc ttctgaaaat agtcctgcag gctagtagat tggttggttg 19140gtttccatgt accagaaggc ttaccctatt agttgaaagt tgaaactttg ttccctactc 19200aattcctagt tgtgtaaatg tatgtatatg taatgtgtat aaaacgtagt acttaaatga

19260ctaggagtgg ttcttgagac cgatgagaga tgggagcaga actaaagatg atgacataat 19320taagaacgaa tttgaaaggc tcttaggttt gaatcctatt cgagaatgtt tttgtcaaag 19380atagtggcga ttttgaacca aagaaaacat ttaaaaaatc agtatccggt tacgttcatg 19440caaatagaaa gtggtctagg atctgattgt aattttagac ttaaagagtc tcttaagatt 19500caatcctggc tgtgtacaaa actacaaata atatatttta gactatttgg ccttaactaa 19560acttccactc attatttact gaggttagag aatagacttg cgaataaaca cattcccgag 19620aaatactcat gatcccataa ttagtcagag ggtatgccaa tcagatctaa gaacacacat 19680tccctcaaat tttaatgcac atgtaatcat agtttagcac aattcaaaaa taatgtagta 19740ttaaagacag aaatttgtag actttttttt ggcgttaaaa gaagactaag tttatacgta 19800cattttattt taagtggaaa accgaaattt tccatcgaaa tatatgaatt tagtatatat 19860atttctgcaa tgtactattt tgctattttg gcaactttca gtggactact actttattac 19920aatgtgtatg gatgcatgag tttgagtata cacatgtcta aatgcatgct ttgtaaaacg 19980taacggacca caaaagagga tccatacaaa tacatctcat agcttcctcc attattttcc 20040gacacaaaca gagcatttta caacaattac caacaacaac aaacaacaaa caacattaca 20100attacattta caattaccat accatggcct ctatcgctat ccctgctgct cttgctggaa 20160ctcttggata cgttacctac aatgtggcta accctgatat cccagcttct gagaaagttc 20220ctgcttactt catgcaggtt gagtactggg gacctactat cggaactatt ggatacctcc 20280tcttcatcta cttcggaaag cgtatcatgc agaacagatc tcaacctttc ggactcaaga 20340acgctatgct cgtttacaac ttctaccaga ccttcttcaa cagctactgc atctaccttt 20400tcgttacttc tcatagggct cagggactta aggtttgggg aaacatccct gatatgactg 20460ctaactcttg gggaatctct caggttatct ggcttcacta caacaacaag tacgttgagc 20520ttctcgacac cttcttcatg gtgatgagga agaagttcga ccagctttct ttccttcaca 20580tctaccacca cactcttctc atctggtcat ggttcgttgt tatgaagctt gagcctgttg 20640gagattgcta cttcggatct tctgttaaca ccttcgtgca cgtgatcatg tactcttact 20700acggacttgc tgctcttgga gttaactgtt tctggaagaa gtacatcacc cagatccaga 20760tgcttcagtt ctgtatctgt gcttctcact ctatctacac cgcttacgtt cagaataccg 20820ctttctggct tccttacctt caactctggg ttatggtgaa catgttcgtt ctcttcgcca 20880acttctaccg taagaggtac aagtctaagg gtgctaagaa gcagtgataa gggccgccgc 20940catgtgacag atcgaaggaa gaaagtgtaa taagacgact ctcactactc gatcgctagt 21000gattgtcatt gttatatata ataatgttat ctttcacaac ttatcgtaat gcatgtgaaa 21060ctataacaca ttaatcctac ttgtcatatg ataacactct ccccatttaa aactcttgtc 21120aatttaaaga tataagattc tttaaatgat taaaaaaaat atattataaa ttcaatcact 21180cctactaata aattattaat tattatttat tgattaaaaa aatacttata ctaatttagt 21240ctgaatagaa taattagatt ctagcctgca gggcggccgc ggatcccatg gagtcaaaga 21300ttcaaataga ggacctaaca gaactcgccg taaagactgg cgaacagttc atacagagtc 21360tcttacgact caatgacaag aagaaaatct tcgtcaacat ggtggagcac gacacacttg 21420tctactccaa aaatatcaaa gatacagtct cagaagacca aagggcaatt gagacttttc 21480aacaaagggt aatatccgga aacctcctcg gattccattg cccagctatc tgtcacttta 21540ttgtgaagat agtggaaaag gaaggtggct cctacaaatg ccatcattgc gataaaggaa 21600aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa agatggaccc ccacccacga 21660ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg 21720atatctccac tgacgtaagg gatgacgcac aatcccacta tccttcgcaa gacccttcct 21780ctatataagg aagttcattt catttggaga gaacacgggg gactgaatta aatatgagcc 21840ctgagaggcg tcctgttgaa atcagacctg ctactgctgc tgatatggct gctgtttgtg 21900atatcgtgaa ccactacatc gagacttcta ccgttaactt cagaactgag cctcaaactc 21960ctcaagagtg gatcgatgat cttgagagac tccaagatag atacccttgg cttgttgctg 22020aggttgaggg tgttgttgct ggaatcgctt acgctggacc ttggaaggct agaaacgctt 22080acgattggac tgttgagtct accgtttacg tttcacacag acatcagaga cttggacttg 22140gatctaccct ttacactcac cttctcaagt ctatggaagc tcagggattc aagtctgttg 22200ttgctgttat cggactccct aacgatcctt ctgttagact tcatgaggct cttggataca 22260ctgctagagg aactcttaga gctgctggat acaagcacgg tggatggcat gatgttggat 22320tctggcaaag agatttcgag cttcctgctc ctcctagacc tgttagacca gttactcaga 22380tctgaatttg cgtgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt 22440tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 22500taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 22560atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 22620cgcggtgtca tctatgttac tagatcacta gtgatgtacg gttaaaacca ccccagtaca 22680ttaaaaacgt ccgcaatgtg ttattaagtt gtctaagcgt caatttgttt acaccacaat 22740atatcctgcc accagccagc caacagctcc ccgaccggca gctcggcaca aaatcaccac 22800tcgatacagg cagcccatca gtcc 22824524809DNAArtificial Sequencepga7- mod_e nucleotide sequence 5tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180tcacgacgtt gtaaaacggg cgcctcgatt aaaaatccca attatatttg gtctaattta 240gtttggtatt gagtaaaaca aattcgaacc aaaccaaaat ataaatatat agtttttata 300tatatgcctt taagactttt tatagaattt tctttaaaaa atatctagaa atatttgcga 360ctcttctggc atgtaatatt tcgttaaata tgaagtgctc catttttatt aactttaaat 420aattggttgt acgatcactt tcttatcaag tgttactaaa atgcgtcaat ctctttgttc 480ttccatattc atatgtcaaa atctatcaaa attcttatat atctttttcg aatttgaagt 540gaaatttcga taatttaaaa ttaaatagaa catatcatta tttaggtatc atattgattt 600ttatacttaa ttactaaatt tggttaactt tgaaagtgta catcaacgaa aaattagtca 660aacgactaaa ataaataaat atcatgtgtt attaagaaaa ttctcctata agaatatttt 720aatagatcat atgtttgtaa aaaaaattaa tttttactaa cacatatatt tacttatcaa 780aaatttgaca aagtaagatt aaaataatat tcatctaaca aaaaaaaaac cagaaaatgc 840tgaaaacccg gcaaaaccga accaatccaa accgatatag ttggtttggt ttgattttga 900tataaaccga accaactcgg tccatttgca cccctaatca taatagcttt aatatttcaa 960gatattatta agttaacgtt gtcaatatcc tggaaatttt gcaaaatgaa tcaagcctat 1020atggctgtaa tatgaattta aaagcagctc gatgtggtgg taatatgtaa tttacttgat 1080tctaaaaaaa tatcccaagt attaataatt tctgctagga agaaggttag ctacgattta 1140cagcaaagcc agaatacaaa gaaccataaa gtgattgaag ctcgaaatat acgaaggaac 1200aaatattttt aaaaaaatac gcaatgactt ggaacaaaag aaagtgatat attttttgtt 1260cttaaacaag catcccctct aaagaatggc agttttcctt tgcatgtaac tattatgctc 1320ccttcgttac aaaaattttg gactactatt gggaacttct tctgaaaata gtggcgcccc 1380gcggaaagct tgctagccaa ttggggccca acgttctcga gtttttctag aaggaaactg 1440aaggcgggaa acgacaatct gctagtggat ctcccagtca cgacgttgta aaacgggcgc 1500cccgcggaaa gcttgcggcc gcggtaccgc ccgttcgact cagatcttcc aaggcctcgt 1560ctccgagtcc gctgcttctc gccgcgccga tcacttctcc gccgccaaca aggcttgtag 1620ttaataggaa tcattcaggg attgtgattc cgggcagtag taattaataa tatagtatta 1680gtatagataa tatgtttcgt ttgggatctt tggaacgttg ctctgttcct tgttgttcat 1740tttaaagctt ttgagggata gttgcagaac tgttcggtga tgcttcatcc tctcaagaac 1800tagatttggg taaagaaaca tccatgcatg gatatggaat gttgttcttc cgattggaga 1860ttattttata aaatttaaaa ttcatgattt aaaaaaacac ataaaaacca caaaattcat 1920gatttattga caatacgata caaaattagc accaccggct actggctcat tacacatttc 1980cccttcccct cattctcact ttgtggcttt attattatta ttattacata tattttaccg 2040ttattatttc acgtcacata agcttgttaa ttaatcatta gtgagccttc tcagcctttc 2100cgttaacgta gtagtgctgt cccaccttat caaggttaga gaaagtagcc ttccaagcac 2160cgtagtaaga gagcaccttg tagttgagtc cccacttctt agcgaaagga acgaatcttc 2220tgctaacctc aggctgtctg aattgaggca tatcagggaa gaggtggtgg ataacctgac 2280agttaaggta tcccataagc cagttcacgt atcctctaga aggatcgata tcaacggtgt 2340gatcaacagc gtagttaacc caagaaaggt gcttatcaga tggaacaaca gggaggtgag 2400tatgagaagt agagaagtga gcgaaaaggt acatgtaagc gatccagttt ccgaaagtga 2460accaccagta agcaacaggc caagagtatc cagtagcaag cttgataaca gcggttctaa 2520caacatgaga aacgagcatc caagaagcct cttcgtagtt cttcttacgg agaacttgtc 2580tagggtggag aacgtagatc cagaaagctt gaacaagaag tccagaggta acaggaacga 2640aagtccaagc ttgaagtcta gcccaagctc tagagaatcc tctaggtctg ttatcctcaa 2700cagcagtgtt gaagaaagcc acagcaggag tggtatcaag atccatatcg tgtctaacct 2760tttgaggggt agcatggtgc ttgttatgca tctggttcca catctcacca gaagtagaaa 2820gtccgaatcc acaagtcata gcctgaagtc tcttgtccac gtaaacagat ccggtaagag 2880agttatgtcc accctcatgt tgaacccatc cacatctagc tccgaagaaa gcaccgtaaa 2940caacagaagc aatgataggg tatccagcgt acataagagc agttccaaga gcgaatgtag 3000caagaagctc gagaagtctg taagccacat gggtgataga aggcttgaag aatccatctc 3060tctcaagctc agcacgccat ctagcgaaat cctcaagcat aggagcatcc tcagactcag 3120atctcttgat ctcagcaggt ctagaaggca aagctctaag catcttccaa gccttgagag 3180aacgcatgtg gaattctttg aaagcctcag tagcatcagc accagtgtta gcaagcatgt 3240agaagatcac agatccacca gggtgcttga agttagtcac atcgtactca acgtcctcaa 3300ctctaaccca tctagtctcg aaagtagcag caagctcatg aggctcaaga gtcttaagat 3360caacaggagc agtagaagca tccttagcat caagagcctc agcagaagat ttagacctgg 3420taagtggaga tctaggagaa gatcttccat cagtcttagg agggcacatg gtatggtaat 3480tgtaaatgta attgtaatgt tgtttgttgt ttgttgttgt tggtaattgt tgtaaaatta 3540attaagtggg tatcttttgg atggataagc aagtagtgat gatgttctag gtgaagtgat 3600gggggtgttt tatagcggga gatggtgaaa tggatggtcg ccacataaga aatggagggg 3660aagggttctt gcgccattct tcagtttgca tggatgcatg ggtttcattt tgtaacacgt 3720aataaggaca atgaagtgca ggtgtctctc aagtttcaga ggggatatgt ggacagaaga 3780agaacggcga tgatattgat ggaaatggcc atctagtgtg aatctattcg gttgataata 3840ctagtgcatt ttggccgtta atcccttcaa ttaactgcac aaacttcagt tgagtattga 3900ttatttgatt ataggttctg taaacacaat accaagttta tttagagggg agacatacaa 3960atagtttcga tataaataat agagtggtta aacttagtta ttaaaactat atataaagtc 4020taaaagttaa attatttttt taattgcaaa tatataaagt ctaaaggggt tacattattt 4080cttaagagat gtaactctgt tggaatctga cttaatccgt ctcatcactc tggtttccag 4140ttctaatcta atgaattgtt ttctgccaaa gaatttgaag caagaagtaa attgatcaat 4200gccgtcaacc cacaccaaac cgtcaaccca ctaccatcgc cgcggagacc cccaaactca 4260acctccaccc atcggtaaga agcacagggc agcccgcacc accaccaatt tggcgtgcat 4320gacacctagg gacttggcac gggaggcggc gcacgtggat gcaaatgacg ggatatcaga 4380tgacaggaaa cgacgttgag agaccatacg atgtagaata tgagctcacc atcaacgaga 4440aactaggaaa atcacaaaaa aaacaactct cgtaattgta cgagtggcac agatgggtct 4500gcctcaacat atctctaata cggcgaagcc tgcccaacac gtagttgccg gaatccggtg 4560tggagctcac gactctgaaa gataggcgct tcctgtttcg tttcgctcac ccactggacg 4620tccgtcatgt gatggatttc ggtcattggt ttgctgacaa ccacattctg aagctccatg 4680agatgagtct tcacaatagg tcctgctcaa taccgtggag ttatggttgc aagtccataa 4740cttgccgttc gaatattttg cggagccagt cggacgggaa ttggcgagct cggctgacac 4800ctataaaggc catgacaaga agaaccaaaa gttcttccct aatgctttca tgaggcttcg 4860ggtcgttatg gatgtcggaa aacccctctt gaaggaacga gacgttatta tgcatgacgg 4920taagactatt acttgtcagt ataagtatga aagattacct gtcttctgct ttgtttgtgg 4980attgattgga cacgttgaaa aaaaatgtgc acttcgattt caatactcag agatcgactt 5040cccttttctc taggagtatt cgatcaaggc attaacatgg aaggaagctc aagctctaaa 5100ggcttcacaa tggaacctga aaaatttcaa caagcctaaa ctgaaatcga agtcaaatca 5160cccaaccggg agctctaaat cagcaaacac tcctcctcca cagtatccaa tcatcgtgca 5220cgatgctcca ggtattgcaa gccaggtatt gcaagctagg agtaggatag agaccttaaa 5280cgtcgttggt gtgaagagtc atcttcagac ctaatggaga tagatgtaga cggcggcacg 5340aagactctga aacaccagaa aggctagtcc aggataagga tctgctatcc caactgacct 5400ctcgttagtc ccaaggcctc tcaactagag caggaggaag gatggtcaca agactaggat 5460aatgatgttt ccaatatgaa cctgaatgtc catagctaat ttttttagtc ttgcttctgc 5520actttttgtt tattatgttc tggtgactat gttatttacc cttgtccgta tgcttgaggg 5580taccctagta gattggttgg ttggtttcca tgtaccagaa ggcttaccct attagttgaa 5640agttgaaact ttgttcccta ctcaattcct agttgtgtaa atgtatgtat atgtaatgtg 5700tataaaacgt agtacttaaa tgactaggag tggttcttga gaccgatgag agatgggagc 5760agaactaaag atgatgacat aattaagaac gaatttgaaa ggctcttagg tttgaatcct 5820attcgagaat gtttttgtca aagatagtgg cgattttgaa ccaaagaaaa catttaaaaa 5880atcagtatcc ggttacgttc atgcaaatag aaagtggtct aggatctgat tgtaatttta 5940gacttaaaga gtctcttaag attcaatcct ggctgtgtac aaaactacaa ataatatatt 6000ttagactatt tggccttaac taaacttcca ctcattattt actgaggtta gagaatagac 6060ttgcgaataa acacattccc gagaaatact catgatccca taattagtca gagggtatgc 6120caatcagatc taagaacaca cattccctca aattttaatg cacatgtaat catagtttag 6180cacaattcaa aaataatgta gtattaaaga cagaaatttg tagacttttt tttggcgtta 6240aaagaagact aagtttatac gtacatttta ttttaagtgg aaaaccgaaa ttttccatcg 6300aaatatatga atttagtata tatatttctg caatgtacta ttttgctatt ttggcaactt 6360tcagtggact actactttat tacaatgtgt atggatgcat gagtttgagt atacacatgt 6420ctaaatgcat gctttgtaaa acgtaacgga ccacaaaaga ggatccatac aaatacatct 6480catagcttcc tccattattt tccgacacaa acagagcatt ttacaacaat taccaacaac 6540aacaaacaac aaacaacatt acaattacat ttacaattac cataccatgg cctctatcgc 6600tatccctgct gctcttgctg gaactcttgg atacgttacc tacaatgtgg ctaaccctga 6660tatcccagct tctgagaaag ttcctgctta cttcatgcag gttgagtact ggggacctac 6720tatcggaact attggatacc tcctcttcat ctacttcgga aagcgtatca tgcagaacag 6780atctcaacct ttcggactca agaacgctat gctcgtttac aacttctacc agaccttctt 6840caacagctac tgcatctacc ttttcgttac ttctcatagg gctcagggac ttaaggtttg 6900gggaaacatc cctgatatga ctgctaactc ttggggaatc tctcaggtta tctggcttca 6960ctacaacaac aagtacgttg agcttctcga caccttcttc atggtgatga ggaagaagtt 7020cgaccagctt tctttccttc acatctacca ccacactctt ctcatctggt catggttcgt 7080tgttatgaag cttgagcctg ttggagattg ctacttcgga tcttctgtta acaccttcgt 7140gcacgtgatc atgtactctt actacggact tgctgctctt ggagttaact gtttctggaa 7200gaagtacatc acccagatcc agatgcttca gttctgtatc tgtgcttctc actctatcta 7260caccgcttac gttcagaata ccgctttctg gcttccttac cttcaactct gggttatggt 7320gaacatgttc gttctcttcg ccaacttcta ccgtaagagg tacaagtcta agggtgctaa 7380gaagcagtga taaggcgcgc ggcgcgccgg gccgccgcca tgtgacagat cgaaggaaga 7440aagtgtaata agacgactct cactactcga tcgctagtga ttgtcattgt tatatataat 7500aatgttatct ttcacaactt atcgtaatgc atgtgaaact ataacacatt aatcctactt 7560gtcatatgat aacactctcc ccatttaaaa ctcttgtcaa tttaaagata taagattctt 7620taaatgatta aaaaaaatat attataaatt caatcactcc tactaataaa ttattaatta 7680ttatttattg attaaaaaaa tacttatact aatttagtct gaatagaata attagattct 7740agtctcatcc ccttttaaac caacttagta aacgtttttt tttttaattt tatgaagtta 7800agtttttacc ttgtttttaa aaagaatcgt tcataagatg ccatgccaga acattagcta 7860cacgttacac atagcatgca gccgcggaga attgtttttc ttcgccactt gtcactccct 7920tcaaacacct aagagcttct ctctcacagc acacacatac aatcacatgc gtgcatgcat 7980tattacacgt gatcgccatg caaatctcct ttatagccta taaattaact catccgcttc 8040actctttact caaaccaaaa ctcatcgata caaacaagat taaaaacata cacgaggatc 8100ttttacaaca attaccaaca acaacaaaca acaaacaaca ttacaattac atttacaatt 8160accataccat gcctccaagg gactcttact cttatgctgc tcctccttct gctcaacttc 8220acgaagttga tactcctcaa gagcacgaca agaaagagct tgttatcgga gatagggctt 8280acgatgttac caacttcgtt aagagacacc ctggtggaaa gatcattgct taccaagttg 8340gaactgatgc taccgatgct tacaagcagt tccatgttag atctgctaag gctgacaaga 8400tgcttaagtc tcttccttct cgtcctgttc acaagggata ctctccaaga agggctgatc 8460ttatcgctga tttccaagag ttcaccaagc aacttgaggc tgagggaatg ttcgagcctt 8520ctcttcctca tgttgcttac agacttgctg aggttatcgc tatgcatgtt gctggtgctg 8580ctcttatctg gcatggatac actttcgctg gaatcgctat gcttggagtt gttcagggaa 8640gatgtggatg gcttatgcat gagggtggac attactctct cactggaaac attgctttcg 8700acagagctat ccaagttgct tgttacggac ttggatgtgg aatgtctggt gcttggtggc 8760gtaaccagca taacaagcac catgctactc ctcaaaagct tcagcacgat gttgatcttg 8820atacccttcc tctcgttgct ttccatgaga gaatcgctgc taaggttaag tctcctgcta 8880tgaaggcttg gctttctatg caagctaagc ttttcgctcc tgttaccact cttcttgttg 8940ctcttggatg gcagctttac cttcatccta gacacatgct caggactaag cactacgatg 9000agcttgctat gctcggaatc agatacggac ttgttggata ccttgctgct aactacggtg 9060ctggatacgt tctcgcttgt taccttcttt acgttcagct tggagctatg tacatcttct 9120gcaacttcgc tgtttctcat actcacctcc ctgttgttga gcctaacgag catgctactt 9180gggttgagta cgctgctaac cacactacta actgttctcc atcttggtgg tgtgattggt 9240ggatgtctta ccttaactac cagatcgagc accaccttta cccttctatg cctcaattca 9300gacaccctaa gatcgctcct agagttaagc agcttttcga gaagcacgga cttcactacg 9360atgttagagg atacttcgag gctatggctg atactttcgc taaccttgat aacgttgccc 9420atgctcctga gaagaaaatg cagtaatgag atcgttcaaa catttggcaa taaagtttct 9480taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg 9540ttaagcacgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga 9600ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact 9660aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcggtcgatt aaaaatccca 9720attatatttg gtctaattta gtttggtatt gagtaaaaca aattcgaacc aaaccaaaat 9780ataaatatat agtttttata tatatgcctt taagactttt tatagaattt tctttaaaaa 9840atatctagaa atatttgcga ctcttctggc atgtaatatt tcgttaaata tgaagtgctc 9900catttttatt aactttaaat aattggttgt acgatcactt tcttatcaag tgttactaaa 9960atgcgtcaat ctctttgttc ttccatattc atatgtcaaa atctatcaaa attcttatat 10020atctttttcg aatttgaagt gaaatttcga taatttaaaa ttaaatagaa catatcatta 10080tttaggtatc atattgattt ttatacttaa ttactaaatt tggttaactt tgaaagtgta 10140catcaacgaa aaattagtca aacgactaaa ataaataaat atcatgtgtt attaagaaaa 10200ttctcctata agaatatttt aatagatcat atgtttgtaa aaaaaattaa tttttactaa 10260cacatatatt tacttatcaa aaatttgaca aagtaagatt aaaataatat tcatctaaca 10320aaaaaaaaac cagaaaatgc tgaaaacccg gcaaaaccga accaatccaa accgatatag 10380ttggtttggt ttgattttga tataaaccga accaactcgg tccatttgca cccctaatca 10440taatagcttt aatatttcaa gatattatta agttaacgtt gtcaatatcc tggaaatttt 10500gcaaaatgaa tcaagcctat atggctgtaa tatgaattta aaagcagctc gatgtggtgg 10560taatatgtaa tttacttgat tctaaaaaaa tatcccaagt attaataatt tctgctagga 10620agaaggttag ctacgattta cagcaaagcc agaatacaaa gaaccataaa gtgattgaag 10680ctcgaaatat acgaaggaac aaatattttt aaaaaaatac gcaatgactt ggaacaaaag 10740aaagtgatat attttttgtt cttaaacaag catcccctct aaagaatggc agttttcctt 10800tgcatgtaac tattatgctc ccttcgttac aaaaattttg gactactatt gggaacttct 10860tctgaaaata gtgatagaac ccacacgagc atgtgctttc catttaattt taaaaaccaa 10920gaaacataca tacataacat tccatcagcc tctctctctt tttattacgg ttaatgactt 10980aaaacacatc ttattatccc atccttaaca cctagcagtg tctttatacg atctcatcga 11040tcaccacttc aaaaccatgc agactgctgc tgcccctgga gctggcatcg gctaggctgg 11100gtgccgcact gtcccggaag gtccctagcg acttgtttag attgatggga ccacctctca 11160acttcctgct gctgtccctg ctgctggatg tcctgcctca tctggccgat tgcacgctcc 11220agtcccctgc atgtgcactc gctcctcaat tgcttaagat catcgcagca gctatcgaag 11280tgctggctct gttgccctcc tccacggcct tggttgtagt agtagctgcc gccgcccttc 11340tggacttttt cccacaggaa ccgccgaata attcgataga accacacgag catgtgcttt

11400catttatttt aaaaaccaag aaacatacat aacatttcat cagcctctct ctctctctct 11460ctctctctct ctctctctct ctctctctct ctctctttat tacagctgtt acactaactt 11520aaaacacatt catctcatta ttattattat tatccatcct taacacctag cagtgtcttt 11580gtacgatctc ataatcgatc accccttcat caggtatcct taggcttcac tccaacgttg 11640ttgcagttac ggaacatgta cacaccatca tggttctcaa cgaactggca agatctccaa 11700gttttccaaa ggctaaccca catgttctca tcggtgtgtc tgtagtgctc tcccataact 11760ttcttgatgc actcggtagc ttctctagca tggtagaatg ggatccttga aacgtagtga 11820tggagcacat gagtctcgat gatgtcatgg aagatgattc cgaggattcc gaactctcta 11880tcgatagtag cagcagcacc cttagcgaaa gtccactctt gagcatcgta atgaggcata 11940gaagaatcgg tgtgctgaag gaaggtaacg aaaacaagcc agtggttaac aaggatccaa 12000ggacagaacc atgtgatgaa agtaggccag aatccgaaaa ccttgtaagc ggtgtaaaca 12060gaagtgaggg tagcaaggat tccaagatca gaaagaacga tgtaccagta gtccttctta 12120tcgaaaacag ggctagaagg ccagtagtga gacttgaaga acttagaaac accagggtaa 12180ggttgtccag tagcgttagt agcaaggtaa agagaaagtc ctccaagctg ttggaacaag 12240agagcgaaaa cagagtagat aggagtttcc tcagcgatat cgtgaaggct ggtaacttgg 12300tgcttctctt tgaattcctc ggcggtgtaa ggaacgaaaa ccatatctct ggtcatgtgt 12360ccagtagcct tatggtgctt agcatgagag aacttccagc tgaagtaagg aaccataaca 12420agagagtgga gaacccatcc aacggtatcg ttaacccatc cgtagttaga gaaagcagaa 12480tgtccacact catgtccaag gatccagatt ccgaatccga aacaagagat agagaacacg 12540taagcagacc aagcagcgaa tctaaggaat tcgttaggga gaagagggat gtaggtaagt 12600ccaacgtaag cgatagcaga gatagccacg atatctctca ccacgtaaga catagacttc 12660acgagagatc tctcgtaaca gtgcttaggg atagcgtcaa ggatatcctt gatggtgtaa 12720tctggcacct tgaaaacgtt tccgaaggta tcgatagcgg tcttttgctg cttgaaagat 12780gcaacgtttc cagaacgcct aacggtctta gtagatccct caaggatctc agatccagac 12840acggtaacct tagacatggt atggtaattg taaatgtaat tgtaatgttg tttgttgttt 12900gttgttgttg gtaattgttg taaaattttt ggtggtgatt ggttctttaa ggtgtgagag 12960tgagttgtga gttgtgtggt gggtttggtg agattgggga tggtgggttt atatagtgga 13020gactgaggaa tggggtcgtg agtgttaact ttgcatgggc tacacgtggg ttcttttggg 13080cttacacgta gtattattca tgcaaatgca gccaatacat atacggtatt ttaataatgt 13140gtgggaatac aatatgccga gtattttact aattttggca atgacaagtg tacatttgga 13200ttatcttact tggcctctct tgctttaatt tggattattt ttattctctt accttggccg 13260ttcatattca catccctaaa ggcaagacag aattgaatgg tggccaaaaa ttaaaacgat 13320ggatatgacc tacatagtgt aggatcaatt aacgtcgaag gaaaatactg attctctcaa 13380gcatacggac aagggtaaat aacatagtca ccagaacata ataaacaaaa agtgcagaag 13440caagactaaa aaaattagct atggacattc aggttcatat tggaaacatc attatcctag 13500tcttgtgacc atccttcctc ctgctctagt tgagaggcct tgggactaac gagaggtcag 13560ttgggatagc agatccttat cctggactag cctttctggt gtttcagagt cttcgtgccg 13620ccgtctacat ctatctccat taggtctgaa gatgactctt cacaccaacg acgtttaagg 13680tctctatcct actcctagct tgcaatacct ggcttgcaat acctggagca tcgtgcacga 13740tgattggata ctgtggagga ggagtgtttg ctgatttaga gctcccggtt gggtgatttg 13800acttcgattt cagtttaggc ttgttgaaat ttttcaggtt ccattgtgaa gcctttagag 13860cttgagcttc cttccatgtt aatgccttga tcgaatactc ctagagaaaa gggaagtcga 13920tctctgagta ttgaaatcga agtgcacatt ttttttcaac gtgtccaatc aatccacaaa 13980caaagcagaa gacaggtaat ctttcatact tatactgaca agtaatagtc ttaccgtcat 14040gcataataac gtctcgttcc ttcaagaggg gttttccgac atccataacg acccgaagcc 14100tcatgaaagc attagggaag aacttttggt tcttcttgtc atggccttta taggtgtcag 14160ccgagctcgc caattcccgt ccgactggct ccgcaaaata ttcgaacggc aagttatgga 14220cttgcaacca taactccacg gtattgagca ggacctattg tgaagactca tctcatggag 14280cttcagaatg tggttgtcag caaaccaatg accgaaatcc atcacatgac ggacgtccag 14340tgggtgagcg aaacgaaaca ggaagcgcct atctttcaga gtcgtgagct ccacaccgga 14400ttccggcaac tacgtgttgg gcaggcttcg ccgtattaga gatatgttga ggcagaccca 14460tctgtgccac tcgtacaatt acgagagttg ttttttttgt gattttccta gtttctcgtt 14520gatggtgagc tcatattcta catcgtatgg tctctcaacg tcgtttcctg tcatctgata 14580tcccgtcatt tgcatccacg tgcgccgcct cccgtgccaa gtccctaggt gtcatgcacg 14640ccaaattggt ggtggtgcgg gctgccctgt gcttcttacc gatgggtgga ggttgagttt 14700gggggtctcc gcggcgatgg tagtgggttg acggtttggt gtgggttgac ggcattgatc 14760aatttacttc ttgcttcaaa ttctttggca gaaaacaatt cattagatta gaactggaaa 14820ccagagtgat gagacggatt aagtcagatt ccaacagagt tacatctctt aagaaataat 14880gtaacccctt tagactttat atatttgcaa ttaaaaaaat aatttaactt ttagacttta 14940tatatagttt taataactaa gtttaaccac tctattattt atatcgaaac tatttgtatg 15000tctcccctct aaataaactt ggtattgtgt ttacagaacc tataatcaaa taatcaatac 15060tcaactgaag tttgtgcagt taattgaagg gattaacggc caaaatgcac tagtattatc 15120aaccgaatag attcacacta gatggccatt tccatcaata tcatcgccgt tcttcttctg 15180tccacatatc ccctctgaaa cttgagagac acctgcactt cattgtcctt attacgtgtt 15240acaaaatgaa acccatgcat ccatgcaaac tgaagaatgg cgcaagaacc cttcccctcc 15300atttcttatg tggcgaccat ccatttcacc atctcccgct ataaaacacc cccatcactt 15360cacctagaac atcatcacta cttgcttatc catccaaaag atacccactt ttacaacaat 15420taccaacaac aacaaacaac aaacaacatt acaattacat ttacaattac cataccatgc 15480cacctagcgc tgctaagcaa atgggagctt ctactggtgt tcatgctggt gttactgact 15540cttctgcttt caccagaaag gatgttgctg atagacctga tctcaccatc gttggagatt 15600ctgtttacga tgctaaggct ttcagatctg agcatcctgg tggtgctcat ttcgtttctt 15660tgttcggagg aagagatgct actgaggctt tcatggaata ccatagaagg gcttggccta 15720agtctagaat gtctagattc cacgttggat ctcttgcttc tactgaggaa cctgttgctg 15780ctgatgaggg ataccttcaa ctttgtgcta ggatcgctaa gatggtgcct tctgtttctt 15840ctggattcgc tcctgcttct tactgggtta aggctggact tatccttgga tctgctatcg 15900ctcttgaggc ttacatgctt tacgctggaa agagacttct cccttctatc gttcttggat 15960ggcttttcgc tcttatcggt cttaacatcc agcatgatgc taaccatggt gctttgtcta 16020agtctgcttc tgttaacctt gctcttggac tttgtcagga ttggatcgga ggatctatga 16080tcctttggct tcaagagcat gttgttatgc accacctcca cactaacgat gttgataagg 16140atcctgatca aaaggctcac ggtgctctta gactcaagcc tactgatgct tggtcaccta 16200tgcattggct tcagcatctt taccttttgc ctggtgagac tatgtacgct ttcaagcttt 16260tgttcctcga catctctgag cttgttatgt ggcgttggga gggtgagcct atctctaagc 16320ttgctggata cctctttatg ccttctttgc ttctcaagct taccttctgg gctagattcg 16380ttgctttgcc tctttacctt gctccttctg ttcatactgc tgtgtgtatc gctgctactg 16440ttatgactgg atctttctac ctcgctttct tcttcttcat ctcccacaac ttcgagggtg 16500ttgcttctgt tggacctgat ggatctatca cttctatgac tagaggtgct agcttcctta 16560agagacaagc tgagacttct tctaacgttg gaggacctct tcttgctact cttaacggtg 16620gactcaacta ccaaattgag catcacttgt tccctagagt tcaccatgga ttctacccta 16680gacttgctcc tcttgttaag gctgagcttg aggctagagg aatcgagtac aagcactacc 16740ctactatctg gtctaacctt gcttctaccc tcagacatat gtacgctctt ggaagaaggc 16800ctagatctaa ggctgagtaa tgacaagctt atgtgacgtg aaataataac ggtaaaatat 16860atgtaataat aataataata aagccacaaa gtgagaatga ggggaagggg aaatgtgtaa 16920tgagccagta gccggtggtg ctaattttgt atcgtattgt caataaatca tgaattttgt 16980ggtttttatg tgttttttta aatcatgaat tttaaatttt ataaaataat ctccaatcgg 17040aagaacaaca ttccatatcc atgcatggat gtttctttac ccaaatctag ttcttgagag 17100gatgaagcat caccgaacag ttctgcaact atccctcaaa agctttaaaa tgaacaacaa 17160ggaacagagc aacgttccaa agatcccaaa cgaaacatat tatctatact aatactatat 17220tattaattac tactgcccgg aatcacaatc cctgaatgat tcctattaac tacaagcctt 17280gttggcggcg gagaagtgat cggcgcggcg agaagcagcg gactcggaga cgaggccttg 17340gaagatctga gtcgaacggg cagaatcagt attttccttc gacgttaatt gatcctacac 17400tatgtaggtc atatccatcg ttttaatttt tggccaccat tcaattctgt cttgccttta 17460gggatgtgaa tatgaacggc caaggtaaga gaataaaaat aatccaaatt aaagcaagag 17520aggccaagta agataatcca aatgtacact tgtcattgcc aaaattagta aaatactcgg 17580catattgtat tcccacacat tattaaaata ccgtatatgt attggctgca tttgcatgaa 17640taatactacg tgtaagccca aaagaaccca cgtgtagccc atgcaaagtt aacactcacg 17700accccattcc tcagtctcca ctatataaac ccaccatccc caatctcacc aaacccacca 17760cacaactcac aactcactct cacaccttaa agaaccaatc accaccaaaa attttacaac 17820aattaccaac aacaacaaac aacaaacaac attacaatta catttacaat taccatacca 17880tgagcgctgt taccgttact ggatctgatc ctaagaacag aggatcttct agcaacaccg 17940agcaagaggt tccaaaagtt gctatcgata ccaacggaaa cgtgttctct gttcctgatt 18000tcaccatcaa ggacatcctt ggagctatcc ctcatgagtg ttacgagaga agattggcta 18060cctctctcta ctacgtgttc agagatatct tctgcatgct taccaccgga taccttaccc 18120ataagatcct ttaccctctc ctcatctctt acacctctaa cagcatcatc aagttcactt 18180tctgggccct ttacacttac gttcaaggac ttttcggaac cggaatctgg gttctcgctc 18240atgagtgtgg acatcaagct ttctctgatt acggaatcgt gaacgatttc gttggatgga 18300cccttcactc ttaccttatg gttccttact tcagctggaa gtactctcat ggaaagcacc 18360ataaggctac tggacacatg accagagata tggttttcgt tcctgccacc aaagaggaat 18420tcaagaagtc taggaacttc ttcggtaacc tcgctgagta ctctgaggat tctccactta 18480gaacccttta cgagcttctt gttcaacaac ttggaggatg gatcgcttac ctcttcgtta 18540acgttacagg acaaccttac cctgatgttc cttcttggaa atggaaccac ttctggctta 18600cctctccact tttcgagcaa agagatgctc tctacatctt cctttctgat cttggaatcc 18660tcacccaggg aatcgttctt actctttggt acaagaaatt cggaggatgg tcccttttca 18720tcaactggtt cgttccttac atctgggtta accactggct cgttttcatc acattccttc 18780agcacactga tcctactatg cctcattaca acgctgagga atggactttc gctaagggtg 18840ctgctgctac tatcgataga aagttcggat tcatcggacc tcacatcttc catgatatca 18900tcgagactca tgtgcttcac cactactgtt ctaggatccc attctacaac gctagacctg 18960cttctgaggc tatcaagaaa gttatgggaa agcactacag gtctagcgac gagaacatgt 19020ggaagtcact ttggaagtct ttcaggtctt gccaatacgt tgacggtgat aacggtgttc 19080tcatgttccg taacatcaac aactgcggag ttggagctgc tgagaagtaa tgaaggggtg 19140atcgattatg agatcgtaca aagacactgc taggtgttaa ggatggataa taataataat 19200aatgagatga atgtgtttta agttagtgta acagctgtaa taaagagaga gagagagaga 19260gagagagaga gagagagaga gagagagaga gagaggctga tgaaatgtta tgtatgtttc 19320ttggttttta aaataaatga aagcacatgc tcgtgtggtt ctatcgaatt attcggcggt 19380tcctgtggga aaaagtccag aagggccgcc gcagctacta ctacaaccaa ggccgtggag 19440gagggcaaca gagccagcac ttcgatagct gctgcgatga tcttaagcaa ttgaggagcg 19500agtgcacatg caggggactg gagcgtgcaa tcggccagat gaggcaggac atccagcagc 19560agggacagca gcaggaagtt gagaggtggt cccatcaatc taaacaagtc gctagggacc 19620ttccgggaca gtgcggcacc cagcctagcc gatgccagct ccaggggcag cagcagtctg 19680catggttttg aagtggtgat cgatgagatc gtataaagac actgctaggt gttaaggatg 19740ggataataag atgtgtttta agtcattaac cgtaataaaa agagagagag gctgatggaa 19800tgttatgtat gtatgtttct tggtttttaa aattaaatgg aaagcacatg ctcgtgtggg 19860ttctatctcg attaaaaatc ccaattatat ttggtctaat ttagtttggt attgagtaaa 19920acaaattcga accaaaccaa aatataaata tatagttttt atatatatgc ctttaagact 19980ttttatagaa ttttctttaa aaaatatcta gaaatatttg cgactcttct ggcatgtaat 20040atttcgttaa atatgaagtg ctccattttt attaacttta aataattggt tgtacgatca 20100ctttcttatc aagtgttact aaaatgcgtc aatctctttg ttcttccata ttcatatgtc 20160aaaatctatc aaaattctta tatatctttt tcgaatttga agtgaaattt cgataattta 20220aaattaaata gaacatatca ttatttaggt atcatattga tttttatact taattactaa 20280atttggttaa ctttgaaagt gtacatcaac gaaaaattag tcaaacgact aaaataaata 20340aatatcatgt gttattaaga aaattctcct ataagaatat tttaatagat catatgtttg 20400taaaaaaaat taatttttac taacacatat atttacttat caaaaatttg acaaagtaag 20460attaaaataa tattcatcta acaaaaaaaa aaccagaaaa tgctgaaaac ccggcaaaac 20520cgaaccaatc caaaccgata tagttggttt ggtttgattt tgatataaac cgaaccaact 20580cggtccattt gcacccctaa tcataatagc tttaatattt caagatatta ttaagttaac 20640gttgtcaata tcctggaaat tttgcaaaat gaatcaagcc tatatggctg taatatgaat 20700ttaaaagcag ctcgatgtgg tggtaatatg taatttactt gattctaaaa aaatatccca 20760agtattaata atttctgcta ggaagaaggt tagctacgat ttacagcaaa gccagaatac 20820aaagaaccat aaagtgattg aagctcgaaa tatacgaagg aacaaatatt tttaaaaaaa 20880tacgcaatga cttggaacaa aagaaagtga tatatttttt gttcttaaac aagcatcccc 20940tctaaagaat ggcagttttc ctttgcatgt aactattatg ctcccttcgt tacaaaaatt 21000ttggactact attgggaact tcttctgaaa atagtcctgc aggctagtag attggttggt 21060tggtttccat gtaccagaag gcttacccta ttagttgaaa gttgaaactt tgttccctac 21120tcaattccta gttgtgtaaa tgtatgtata tgtaatgtgt ataaaacgta gtacttaaat 21180gactaggagt ggttcttgag accgatgaga gatgggagca gaactaaaga tgatgacata 21240attaagaacg aatttgaaag gctcttaggt ttgaatccta ttcgagaatg tttttgtcaa 21300agatagtggc gattttgaac caaagaaaac atttaaaaaa tcagtatccg gttacgttca 21360tgcaaataga aagtggtcta ggatctgatt gtaattttag acttaaagag tctcttaaga 21420ttcaatcctg gctgtgtaca aaactacaaa taatatattt tagactattt ggccttaact 21480aaacttccac tcattattta ctgaggttag agaatagact tgcgaataaa cacattcccg 21540agaaatactc atgatcccat aattagtcag agggtatgcc aatcagatct aagaacacac 21600attccctcaa attttaatgc acatgtaatc atagtttagc acaattcaaa aataatgtag 21660tattaaagac agaaatttgt agactttttt ttggcgttaa aagaagacta agtttatacg 21720tacattttat tttaagtgga aaaccgaaat tttccatcga aatatatgaa tttagtatat 21780atatttctgc aatgtactat tttgctattt tggcaacttt cagtggacta ctactttatt 21840acaatgtgta tggatgcatg agtttgagta tacacatgtc taaatgcatg ctttgtaaaa 21900cgtaacggac cacaaaagag gatccataca aatacatctc atagcttcct ccattatttt 21960ccgacacaaa cagagcattt tacaacaatt accaacaaca acaaacaaca aacaacatta 22020caattacatt tacaattacc ataccatgga atttgctcaa cctctcgttg ctatggctca 22080agagcagtac gctgctatcg atgctgttgt tgctcctgct atcttctctg ctaccgactc 22140tattggatgg ggactcaagc ctatctcttc tgctactaag gatctccctc tcgttgaatc 22200tcctacccct cttatccttt ctctcctcgc ttacttcgct atcgttggtt ctggactcgt 22260ttaccgtaaa gtgttcccta gaaccgttaa gggacaggat cctttccttc tcaaggctct 22320tatgctcgct cacaacgttt tccttatcgg actcagcctt tacatgtgcc tcaagctcgt 22380ttacgaggct tacgtgaaca agtactcctt ctggggaaac gcttacaacc ctgctcaaac 22440cgagatggct aaggtgatct ggatcttcta cgtgtccaag atctacgagt tcatggacac 22500cttcatcatg cttctcaagg gaaacgttaa ccaggtttcc ttcctccatg tttaccacca 22560cggatctatc tctggaatct ggtggatgat cacttatgct gctccaggtg gagatgctta 22620cttctctgct gctctcaact cttgggttca tgtgtgcatg tacacctact acttcatggc 22680tgctgttctt cctaaggacg aaaagaccaa gagaaagtac ctttggtggg gaagatacct 22740tacccagatg caaatgttcc agttcttcat gaaccttctc caggctgttt acctcctcta 22800ctcttcttct ccttacccta agttcattgc tcaactcctc gttgtttaca tggttaccct 22860cctcatgctt ttcggaaact tctactacat gaagcaccac gcttctaagt gataagggcc 22920gccgccatgt gacagatcga aggaagaaag tgtaataaga cgactctcac tactcgatcg 22980ctagtgattg tcattgttat atataataat gttatctttc acaacttatc gtaatgcatg 23040tgaaactata acacattaat cctacttgtc atatgataac actctcccca tttaaaactc 23100ttgtcaattt aaagatataa gattctttaa atgattaaaa aaaatatatt ataaattcaa 23160tcactcctac taataaatta ttaattatta tttattgatt aaaaaaatac ttatactaat 23220ttagtctgaa tagaataatt agattctagc ctgcagggcg gccgcggatc ccatggagtc 23280aaagattcaa atagaggacc taacagaact cgccgtaaag actggcgaac agttcataca 23340gagtctctta cgactcaatg acaagaagaa aatcttcgtc aacatggtgg agcacgacac 23400acttgtctac tccaaaaata tcaaagatac agtctcagaa gaccaaaggg caattgagac 23460ttttcaacaa agggtaatat ccggaaacct cctcggattc cattgcccag ctatctgtca 23520ctttattgtg aagatagtgg aaaaggaagg tggctcctac aaatgccatc attgcgataa 23580aggaaaggcc atcgttgaag atgcctctgc cgacagtggt cccaaagatg gacccccacc 23640cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg 23700atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt cgcaagaccc 23760ttcctctata taaggaagtt catttcattt ggagagaaca cgggggactg aattaaatat 23820gagccctgag aggcgtcctg ttgaaatcag acctgctact gctgctgata tggctgctgt 23880ttgtgatatc gtgaaccact acatcgagac ttctaccgtt aacttcagaa ctgagcctca 23940aactcctcaa gagtggatcg atgatcttga gagactccaa gatagatacc cttggcttgt 24000tgctgaggtt gagggtgttg ttgctggaat cgcttacgct ggaccttgga aggctagaaa 24060cgcttacgat tggactgttg agtctaccgt ttacgtttca cacagacatc agagacttgg 24120acttggatct accctttaca ctcaccttct caagtctatg gaagctcagg gattcaagtc 24180tgttgttgct gttatcggac tccctaacga tccttctgtt agacttcatg aggctcttgg 24240atacactgct agaggaactc ttagagctgc tggatacaag cacggtggat ggcatgatgt 24300tggattctgg caaagagatt tcgagcttcc tgctcctcct agacctgtta gaccagttac 24360tcagatctga atttgcgtga tcgttcaaac atttggcaat aaagtttctt aagattgaat 24420cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta 24480ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg 24540caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta 24600tcgcgcgcgg tgtcatctat gttactagat cactagtgat gtacggttaa aaccacccca 24660gtacattaaa aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc 24720acaatatatc ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc 24780accactcgat acaggcagcc catcagtcc 24809626543DNAArtificial SequencepGA7- mod_F nucleotide sequence 6tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180tcacgacgtt gtaaaacggg cgggcggccg cctagaatct aattattcta ttcagactaa 240attagtataa gtattttttt aatcaataaa tattaattaa taatttatta gtaggagtga 300ttgaatttat aatatatttt ttttaatcat ttaaagaatc ttatatcttt aaattgacaa 360gagttttaaa tggggagagt gttatcatat gacaagtagg attaatgtgt tatagtttca 420catgcattac gataagttgt gaaagataac attattatat ataacaatga caatcactag 480cgatcgagta gtgagagtcg tcttattaca ctttcttcct tcgatctgtc acatggcggc 540ggcccgcgat cgcgataatt ctcagtgcgc cttctccgcc ttgccgttga cgtagtagtg 600ctgcccgacc ttatccaagt tcgagaacgt cgccttccag gcgccgtaat aggacagcac 660cttgtagttc agcccccact tcttcgcgaa cgggacgaac cgccggctca cctccggctg 720gcgaaactgc ggcatgtccg ggaacaggtg atgaatgacc tggcagttca gatatcccat 780caaccagttc acgtacccgc gcgacgggtc gatgtccacg gtgtgatcga ccgcgtagtt 840cacccagctc aggtgcttat ccgagggcac gaccgggagg tgcgtgtggc tcgtggagaa 900gtgcgcgaag aggtacatgt acgcgatcca gttgccgaag gtgaaccacc agtacgcgac 960gggccacgag taccccgtcg cgagtttaat caccgcggtc ctgacgacgt gagagacgag 1020catccacgac gcctcctcgt agttcttctt tcgcaacacc tgccgcgggt gcaggacgta 1080gatccagaac gcctggacga gcagcccgga ggtcaccggg acgaacgtcc acgcctgaag 1140ccgagcccac gcgcgggaga accccctcgg ccggttgtcc tccacggcgg tgttaaaaaa 1200cgccaccgcg ggggtcgtgt ccaggtccat gtcgtgcctc actttctgcg gcgtcgcgtg 1260gtgcttattg tgcatctggt tccacatctc cccgctcgtg gacagcccga acccgcacgt 1320catcgcttgg aggcgcttgt cgacgtagac ggaccccgtg agcgagttgt gcccgccctc 1380gtgctggacc caaccgcacc gagcgccgaa gaacgcgccg tacacgacgg acgcgatgat 1440cgggtacccg gcgtacatga gggcggtgcc gagggcgaag gtcgcgagga gctcgagtaa 1500ccgatacgcg acgtgcgtta tcgagggctt aaagaacccg tcgcgttcga gctccgcgcg

1560ccaccgcgcg aaatcctcca acatcggcgc gtcctcgctc tcgctgcgtt tgatctccgc 1620ggggcgcgac ggcagcgctc tgagcatctt ccacgcctta agcgatcgca tgtggaactc 1680cttgaacgcc tccgtggcgt ccgcgcccgt gttcgcgagc atgtagaata tcacgctgcc 1740tcccgggtgt ttgaagtttg tgacgtcgta ctcgacgtcc tccacgcgca cccatcgcgt 1800ctcgaacgtc gccgcgagct cgtgcggctc gagcgttttg agatcgacgg gcgcggtcga 1860cgcgtccttg gcgtcgagcg cctccgcgga ggatttgctg cgcgtcagcg gcgatcgcgg 1920ggacgatcgg ccgtccgtct tcggcgggca catcgtcgcg cgcgcgactt aaaccgacga 1980cggacggacg aacctgcaac ggcgaattat caattgacgc gttgctctgt ttgtgtcgga 2040aaataatgga ggaagctatg agatgtattt gcatggatcc tcttttgtgg tccgttacgt 2100tttgcaaagc atgcatttag acatgtgtat actcaaactc atgcatccat acacattgta 2160ataaagtagt agtccactga aagttgccaa aatagcaaaa tagtacattg cagaaatata 2220tatactaaat tcatatattt cgatggaaaa tttcggtttt ccacttaaaa taaaatgtac 2280gtataaactt agtcttcctt taacgccaaa aaaaagtcta caaatttctg tctttaatac 2340tacattattt ttgaattgtg ctaaactatg attacatgtg cattaaaatt tgagggaatg 2400tgtgttctta gatctgattg gcataccctc tgactaatta tgggatcatg agtatttctc 2460gggaatgtgt ttattcgcaa gtctattctc taacctcagt aaataatgag tggaagttta 2520gttaaggcca aatagtctaa aatatattat ttgtagtttt gtacacagcc aggattgaat 2580cttaagagac tctttaagtc taaaattaca atcagatcct agaccacttt ctatttgcat 2640gaacgtaacc ggatactgat tttttaaatg ttttctttgg ttcaaaatcg ccactatctt 2700tgacaaaaac attctcgaat aggattcaaa cctaagagcc tttcaaattc gttcttaatt 2760atgtcatcat ctttagttct gctcccatct ctcatcggtc tcaagaacca ctcctagtca 2820tttaagtact acgttttata cgcattacat atacatacat ttacacaact aggaattgag 2880tagggaacaa agtttcaact ttcaactaat agggtaagcc ttctggtaca tggaaaccaa 2940ccaaccaatc tactaggcgg ccgcccgtcg ggatcttctg caagcatctc tatttcctga 3000aggtctaacc tcgaagattt aagatttaat tacgtttata attacaaaat tgattctagt 3060atctttaatt taatgcttat acattattaa ttaatttagt actttcaatt tgttttcaga 3120aattatttta ctatttttta taaaataaaa gggagaaaat ggctatttaa actgaaggcg 3180ggaaacgaca atctgctagt ggatctccca gtcacgacgt tgtaaaacgg gcgccccgcg 3240gaaagcttgc ggccgcggta ccgcccgttc gactcagatc ttccaaggcc tcgtctccga 3300gtccgctgct tctcgccgcg ccgatcactt ctccgccgcc aacaaggctt gtagttaata 3360ggaatcattc agggattgtg attccgggca gtagtaatta ataatatagt attagtatag 3420ataatatgtt tcgtttggga tctttggaac gttgctctgt tccttgttgt tcattttaaa 3480gcttttgagg gatagttgca gaactgttcg gtgatgcttc atcctctcaa gaactagatt 3540tgggtaaaga aacatccatg catggatatg gaatgttgtt cttccgattg gagattattt 3600tataaaattt aaaattcatg atttaaaaaa acacataaaa accacaaaat tcatgattta 3660ttgacaatac gatacaaaat tagcaccacc ggctactggc tcattacaca tttccccttc 3720ccctcattct cactttgtgg ctttattatt attattatta catatatttt accgttatta 3780tttcacgtca cataagcttg ttaattaatc attagtgagc cttctcagcc tttccgttaa 3840cgtagtagtg ctgtcccacc ttatcaaggt tagagaaagt agccttccaa gcaccgtagt 3900aagagagcac cttgtagttg agtccccact tcttagcgaa aggaacgaat cttctgctaa 3960cctcaggctg tctgaattga ggcatatcag ggaagaggtg gtggataacc tgacagttaa 4020ggtatcccat aagccagttc acgtatcctc tagaaggatc gatatcaacg gtgtgatcaa 4080cagcgtagtt aacccaagaa aggtgcttat cagatggaac aacagggagg tgagtatgag 4140aagtagagaa gtgagcgaaa aggtacatgt aagcgatcca gtttccgaaa gtgaaccacc 4200agtaagcaac aggccaagag tatccagtag caagcttgat aacagcggtt ctaacaacat 4260gagaaacgag catccaagaa gcctcttcgt agttcttctt acggagaact tgtctagggt 4320ggagaacgta gatccagaaa gcttgaacaa gaagtccaga ggtaacagga acgaaagtcc 4380aagcttgaag tctagcccaa gctctagaga atcctctagg tctgttatcc tcaacagcag 4440tgttgaagaa agccacagca ggagtggtat caagatccat atcgtgtcta accttttgag 4500gggtagcatg gtgcttgtta tgcatctggt tccacatctc accagaagta gaaagtccga 4560atccacaagt catagcctga agtctcttgt ccacgtaaac agatccggta agagagttat 4620gtccaccctc atgttgaacc catccacatc tagctccgaa gaaagcaccg taaacaacag 4680aagcaatgat agggtatcca gcgtacataa gagcagttcc aagagcgaat gtagcaagaa 4740gctcgagaag tctgtaagcc acatgggtga tagaaggctt gaagaatcca tctctctcaa 4800gctcagcacg ccatctagcg aaatcctcaa gcataggagc atcctcagac tcagatctct 4860tgatctcagc aggtctagaa ggcaaagctc taagcatctt ccaagccttg agagaacgca 4920tgtggaattc tttgaaagcc tcagtagcat cagcaccagt gttagcaagc atgtagaaga 4980tcacagatcc accagggtgc ttgaagttag tcacatcgta ctcaacgtcc tcaactctaa 5040cccatctagt ctcgaaagta gcagcaagct catgaggctc aagagtctta agatcaacag 5100gagcagtaga agcatcctta gcatcaagag cctcagcaga agatttagac ctggtaagtg 5160gagatctagg agaagatctt ccatcagtct taggagggca catggtatgg taattgtaaa 5220tgtaattgta atgttgtttg ttgtttgttg ttgttggtaa ttgttgtaaa attaattaag 5280tgggtatctt ttggatggat aagcaagtag tgatgatgtt ctaggtgaag tgatgggggt 5340gttttatagc gggagatggt gaaatggatg gtcgccacat aagaaatgga ggggaagggt 5400tcttgcgcca ttcttcagtt tgcatggatg catgggtttc attttgtaac acgtaataag 5460gacaatgaag tgcaggtgtc tctcaagttt cagaggggat atgtggacag aagaagaacg 5520gcgatgatat tgatggaaat ggccatctag tgtgaatcta ttcggttgat aatactagtg 5580cattttggcc gttaatccct tcaattaact gcacaaactt cagttgagta ttgattattt 5640gattataggt tctgtaaaca caataccaag tttatttaga ggggagacat acaaatagtt 5700tcgatataaa taatagagtg gttaaactta gttattaaaa ctatatataa agtctaaaag 5760ttaaattatt tttttaattg caaatatata aagtctaaag gggttacatt atttcttaag 5820agatgtaact ctgttggaat ctgacttaat ccgtctcatc actctggttt ccagttctaa 5880tctaatgaat tgttttctgc caaagaattt gaagcaagaa gtaaattgat caatgccgtc 5940aacccacacc aaaccgtcaa cccactacca tcgccgcgga gacccccaaa ctcaacctcc 6000acccatcggt aagaagcaca gggcagcccg caccaccacc aatttggcgt gcatgacacc 6060tagggacttg gcacgggagg cggcgcacgt ggatgcaaat gacgggatat cagatgacag 6120gaaacgacgt tgagagacca tacgatgtag aatatgagct caccatcaac gagaaactag 6180gaaaatcaca aaaaaaacaa ctctcgtaat tgtacgagtg gcacagatgg gtctgcctca 6240acatatctct aatacggcga agcctgccca acacgtagtt gccggaatcc ggtgtggagc 6300tcacgactct gaaagatagg cgcttcctgt ttcgtttcgc tcacccactg gacgtccgtc 6360atgtgatgga tttcggtcat tggtttgctg acaaccacat tctgaagctc catgagatga 6420gtcttcacaa taggtcctgc tcaataccgt ggagttatgg ttgcaagtcc ataacttgcc 6480gttcgaatat tttgcggagc cagtcggacg ggaattggcg agctcggctg acacctataa 6540aggccatgac aagaagaacc aaaagttctt ccctaatgct ttcatgaggc ttcgggtcgt 6600tatggatgtc ggaaaacccc tcttgaagga acgagacgtt attatgcatg acggtaagac 6660tattacttgt cagtataagt atgaaagatt acctgtcttc tgctttgttt gtggattgat 6720tggacacgtt gaaaaaaaat gtgcacttcg atttcaatac tcagagatcg acttcccttt 6780tctctaggag tattcgatca aggcattaac atggaaggaa gctcaagctc taaaggcttc 6840acaatggaac ctgaaaaatt tcaacaagcc taaactgaaa tcgaagtcaa atcacccaac 6900cgggagctct aaatcagcaa acactcctcc tccacagtat ccaatcatcg tgcacgatgc 6960tccaggtatt gcaagccagg tattgcaagc taggagtagg atagagacct taaacgtcgt 7020tggtgtgaag agtcatcttc agacctaatg gagatagatg tagacggcgg cacgaagact 7080ctgaaacacc agaaaggcta gtccaggata aggatctgct atcccaactg acctctcgtt 7140agtcccaagg cctctcaact agagcaggag gaaggatggt cacaagacta ggataatgat 7200gtttccaata tgaacctgaa tgtccatagc taattttttt agtcttgctt ctgcactttt 7260tgtttattat gttctggtga ctatgttatt tacccttgtc cgtatgcttg agggtaccct 7320agtagattgg ttggttggtt tccatgtacc agaaggctta ccctattagt tgaaagttga 7380aactttgttc cctactcaat tcctagttgt gtaaatgtat gtatatgtaa tgtgtataaa 7440acgtagtact taaatgacta ggagtggttc ttgagaccga tgagagatgg gagcagaact 7500aaagatgatg acataattaa gaacgaattt gaaaggctct taggtttgaa tcctattcga 7560gaatgttttt gtcaaagata gtggcgattt tgaaccaaag aaaacattta aaaaatcagt 7620atccggttac gttcatgcaa atagaaagtg gtctaggatc tgattgtaat tttagactta 7680aagagtctct taagattcaa tcctggctgt gtacaaaact acaaataata tattttagac 7740tatttggcct taactaaact tccactcatt atttactgag gttagagaat agacttgcga 7800ataaacacat tcccgagaaa tactcatgat cccataatta gtcagagggt atgccaatca 7860gatctaagaa cacacattcc ctcaaatttt aatgcacatg taatcatagt ttagcacaat 7920tcaaaaataa tgtagtatta aagacagaaa tttgtagact tttttttggc gttaaaagaa 7980gactaagttt atacgtacat tttattttaa gtggaaaacc gaaattttcc atcgaaatat 8040atgaatttag tatatatatt tctgcaatgt actattttgc tattttggca actttcagtg 8100gactactact ttattacaat gtgtatggat gcatgagttt gagtatacac atgtctaaat 8160gcatgctttg taaaacgtaa cggaccacaa aagaggatcc atacaaatac atctcatagc 8220ttcctccatt attttccgac acaaacagag cattttacaa caattaccaa caacaacaaa 8280caacaaacaa cattacaatt acatttacaa ttaccatacc atggcctcta tcgctatccc 8340tgctgctctt gctggaactc ttggatacgt tacctacaat gtggctaacc ctgatatccc 8400agcttctgag aaagttcctg cttacttcat gcaggttgag tactggggac ctactatcgg 8460aactattgga tacctcctct tcatctactt cggaaagcgt atcatgcaga acagatctca 8520acctttcgga ctcaagaacg ctatgctcgt ttacaacttc taccagacct tcttcaacag 8580ctactgcatc taccttttcg ttacttctca tagggctcag ggacttaagg tttggggaaa 8640catccctgat atgactgcta actcttgggg aatctctcag gttatctggc ttcactacaa 8700caacaagtac gttgagcttc tcgacacctt cttcatggtg atgaggaaga agttcgacca 8760gctttctttc cttcacatct accaccacac tcttctcatc tggtcatggt tcgttgttat 8820gaagcttgag cctgttggag attgctactt cggatcttct gttaacacct tcgtgcacgt 8880gatcatgtac tcttactacg gacttgctgc tcttggagtt aactgtttct ggaagaagta 8940catcacccag atccagatgc ttcagttctg tatctgtgct tctcactcta tctacaccgc 9000ttacgttcag aataccgctt tctggcttcc ttaccttcaa ctctgggtta tggtgaacat 9060gttcgttctc ttcgccaact tctaccgtaa gaggtacaag tctaagggtg ctaagaagca 9120gtgataaggc gcgcggcgcg ccgggccgcc gccatgtgac agatcgaagg aagaaagtgt 9180aataagacga ctctcactac tcgatcgcta gtgattgtca ttgttatata taataatgtt 9240atctttcaca acttatcgta atgcatgtga aactataaca cattaatcct acttgtcata 9300tgataacact ctccccattt aaaactcttg tcaatttaaa gatataagat tctttaaatg 9360attaaaaaaa atatattata aattcaatca ctcctactaa taaattatta attattattt 9420attgattaaa aaaatactta tactaattta gtctgaatag aataattaga ttctagtctc 9480atcccctttt aaaccaactt agtaaacgtt ttttttttta attttatgaa gttaagtttt 9540taccttgttt ttaaaaagaa tcgttcataa gatgccatgc cagaacatta gctacacgtt 9600acacatagca tgcagccgcg gagaattgtt tttcttcgcc acttgtcact cccttcaaac 9660acctaagagc ttctctctca cagcacacac atacaatcac atgcgtgcat gcattattac 9720acgtgatcgc catgcaaatc tcctttatag cctataaatt aactcatccg cttcactctt 9780tactcaaacc aaaactcatc gatacaaaca agattaaaaa catacacgag gatcttttac 9840aacaattacc aacaacaaca aacaacaaac aacattacaa ttacatttac aattaccata 9900ccatgcctcc aagggactct tactcttatg ctgctcctcc ttctgctcaa cttcacgaag 9960ttgatactcc tcaagagcac gacaagaaag agcttgttat cggagatagg gcttacgatg 10020ttaccaactt cgttaagaga caccctggtg gaaagatcat tgcttaccaa gttggaactg 10080atgctaccga tgcttacaag cagttccatg ttagatctgc taaggctgac aagatgctta 10140agtctcttcc ttctcgtcct gttcacaagg gatactctcc aagaagggct gatcttatcg 10200ctgatttcca agagttcacc aagcaacttg aggctgaggg aatgttcgag ccttctcttc 10260ctcatgttgc ttacagactt gctgaggtta tcgctatgca tgttgctggt gctgctctta 10320tctggcatgg atacactttc gctggaatcg ctatgcttgg agttgttcag ggaagatgtg 10380gatggcttat gcatgagggt ggacattact ctctcactgg aaacattgct ttcgacagag 10440ctatccaagt tgcttgttac ggacttggat gtggaatgtc tggtgcttgg tggcgtaacc 10500agcataacaa gcaccatgct actcctcaaa agcttcagca cgatgttgat cttgataccc 10560ttcctctcgt tgctttccat gagagaatcg ctgctaaggt taagtctcct gctatgaagg 10620cttggctttc tatgcaagct aagcttttcg ctcctgttac cactcttctt gttgctcttg 10680gatggcagct ttaccttcat cctagacaca tgctcaggac taagcactac gatgagcttg 10740ctatgctcgg aatcagatac ggacttgttg gataccttgc tgctaactac ggtgctggat 10800acgttctcgc ttgttacctt ctttacgttc agcttggagc tatgtacatc ttctgcaact 10860tcgctgtttc tcatactcac ctccctgttg ttgagcctaa cgagcatgct acttgggttg 10920agtacgctgc taaccacact actaactgtt ctccatcttg gtggtgtgat tggtggatgt 10980cttaccttaa ctaccagatc gagcaccacc tttacccttc tatgcctcaa ttcagacacc 11040ctaagatcgc tcctagagtt aagcagcttt tcgagaagca cggacttcac tacgatgtta 11100gaggatactt cgaggctatg gctgatactt tcgctaacct tgataacgtt gcccatgctc 11160ctgagaagaa aatgcagtaa tgagatcgtt caaacatttg gcaataaagt ttcttaagat 11220tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc 11280acgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag 11340tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata 11400aattatcgcg cgcggtgtca tctatgttac tagatcggtc gattaaaaat cccaattata 11460tttggtctaa tttagtttgg tattgagtaa aacaaattcg aaccaaacca aaatataaat 11520atatagtttt tatatatatg cctttaagac tttttataga attttcttta aaaaatatct 11580agaaatattt gcgactcttc tggcatgtaa tatttcgtta aatatgaagt gctccatttt 11640tattaacttt aaataattgg ttgtacgatc actttcttat caagtgttac taaaatgcgt 11700caatctcttt gttcttccat attcatatgt caaaatctat caaaattctt atatatcttt 11760ttcgaatttg aagtgaaatt tcgataattt aaaattaaat agaacatatc attatttagg 11820tatcatattg atttttatac ttaattacta aatttggtta actttgaaag tgtacatcaa 11880cgaaaaatta gtcaaacgac taaaataaat aaatatcatg tgttattaag aaaattctcc 11940tataagaata ttttaataga tcatatgttt gtaaaaaaaa ttaattttta ctaacacata 12000tatttactta tcaaaaattt gacaaagtaa gattaaaata atattcatct aacaaaaaaa 12060aaaccagaaa atgctgaaaa cccggcaaaa ccgaaccaat ccaaaccgat atagttggtt 12120tggtttgatt ttgatataaa ccgaaccaac tcggtccatt tgcaccccta atcataatag 12180ctttaatatt tcaagatatt attaagttaa cgttgtcaat atcctggaaa ttttgcaaaa 12240tgaatcaagc ctatatggct gtaatatgaa tttaaaagca gctcgatgtg gtggtaatat 12300gtaatttact tgattctaaa aaaatatccc aagtattaat aatttctgct aggaagaagg 12360ttagctacga tttacagcaa agccagaata caaagaacca taaagtgatt gaagctcgaa 12420atatacgaag gaacaaatat ttttaaaaaa atacgcaatg acttggaaca aaagaaagtg 12480atatattttt tgttcttaaa caagcatccc ctctaaagaa tggcagtttt cctttgcatg 12540taactattat gctcccttcg ttacaaaaat tttggactac tattgggaac ttcttctgaa 12600aatagtgata gaacccacac gagcatgtgc tttccattta attttaaaaa ccaagaaaca 12660tacatacata acattccatc agcctctctc tctttttatt acggttaatg acttaaaaca 12720catcttatta tcccatcctt aacacctagc agtgtcttta tacgatctca tcgatcacca 12780cttcaaaacc atgcagactg ctgctgcccc tggagctggc atcggctagg ctgggtgccg 12840cactgtcccg gaaggtccct agcgacttgt ttagattgat gggaccacct ctcaacttcc 12900tgctgctgtc cctgctgctg gatgtcctgc ctcatctggc cgattgcacg ctccagtccc 12960ctgcatgtgc actcgctcct caattgctta agatcatcgc agcagctatc gaagtgctgg 13020ctctgttgcc ctcctccacg gccttggttg tagtagtagc tgccgccgcc cttctggact 13080ttttcccaca ggaaccgccg aataattcga tagaaccaca cgagcatgtg ctttcattta 13140ttttaaaaac caagaaacat acataacatt tcatcagcct ctctctctct ctctctctct 13200ctctctctct ctctctctct ctctctctct ttattacagc tgttacacta acttaaaaca 13260cattcatctc attattatta ttattatcca tccttaacac ctagcagtgt ctttgtacga 13320tctcataatc gatcacccct tcatcaggta tccttaggct tcactccaac gttgttgcag 13380ttacggaaca tgtacacacc atcatggttc tcaacgaact ggcaagatct ccaagttttc 13440caaaggctaa cccacatgtt ctcatcggtg tgtctgtagt gctctcccat aactttcttg 13500atgcactcgg tagcttctct agcatggtag aatgggatcc ttgaaacgta gtgatggagc 13560acatgagtct cgatgatgtc atggaagatg attccgagga ttccgaactc tctatcgata 13620gtagcagcag cacccttagc gaaagtccac tcttgagcat cgtaatgagg catagaagaa 13680tcggtgtgct gaaggaaggt aacgaaaaca agccagtggt taacaaggat ccaaggacag 13740aaccatgtga tgaaagtagg ccagaatccg aaaaccttgt aagcggtgta aacagaagtg 13800agggtagcaa ggattccaag atcagaaaga acgatgtacc agtagtcctt cttatcgaaa 13860acagggctag aaggccagta gtgagacttg aagaacttag aaacaccagg gtaaggttgt 13920ccagtagcgt tagtagcaag gtaaagagaa agtcctccaa gctgttggaa caagagagcg 13980aaaacagagt agataggagt ttcctcagcg atatcgtgaa ggctggtaac ttggtgcttc 14040tctttgaatt cctcggcggt gtaaggaacg aaaaccatat ctctggtcat gtgtccagta 14100gccttatggt gcttagcatg agagaacttc cagctgaagt aaggaaccat aacaagagag 14160tggagaaccc atccaacggt atcgttaacc catccgtagt tagagaaagc agaatgtcca 14220cactcatgtc caaggatcca gattccgaat ccgaaacaag agatagagaa cacgtaagca 14280gaccaagcag cgaatctaag gaattcgtta gggagaagag ggatgtaggt aagtccaacg 14340taagcgatag cagagatagc cacgatatct ctcaccacgt aagacataga cttcacgaga 14400gatctctcgt aacagtgctt agggatagcg tcaaggatat ccttgatggt gtaatctggc 14460accttgaaaa cgtttccgaa ggtatcgata gcggtctttt gctgcttgaa agatgcaacg 14520tttccagaac gcctaacggt cttagtagat ccctcaagga tctcagatcc agacacggta 14580accttagaca tggtatggta attgtaaatg taattgtaat gttgtttgtt gtttgttgtt 14640gttggtaatt gttgtaaaat ttttggtggt gattggttct ttaaggtgtg agagtgagtt 14700gtgagttgtg tggtgggttt ggtgagattg gggatggtgg gtttatatag tggagactga 14760ggaatggggt cgtgagtgtt aactttgcat gggctacacg tgggttcttt tgggcttaca 14820cgtagtatta ttcatgcaaa tgcagccaat acatatacgg tattttaata atgtgtggga 14880atacaatatg ccgagtattt tactaatttt ggcaatgaca agtgtacatt tggattatct 14940tacttggcct ctcttgcttt aatttggatt atttttattc tcttaccttg gccgttcata 15000ttcacatccc taaaggcaag acagaattga atggtggcca aaaattaaaa cgatggatat 15060gacctacata gtgtaggatc aattaacgtc gaaggaaaat actgattctc tcaagcatac 15120ggacaagggt aaataacata gtcaccagaa cataataaac aaaaagtgca gaagcaagac 15180taaaaaaatt agctatggac attcaggttc atattggaaa catcattatc ctagtcttgt 15240gaccatcctt cctcctgctc tagttgagag gccttgggac taacgagagg tcagttggga 15300tagcagatcc ttatcctgga ctagcctttc tggtgtttca gagtcttcgt gccgccgtct 15360acatctatct ccattaggtc tgaagatgac tcttcacacc aacgacgttt aaggtctcta 15420tcctactcct agcttgcaat acctggcttg caatacctgg agcatcgtgc acgatgattg 15480gatactgtgg aggaggagtg tttgctgatt tagagctccc ggttgggtga tttgacttcg 15540atttcagttt aggcttgttg aaatttttca ggttccattg tgaagccttt agagcttgag 15600cttccttcca tgttaatgcc ttgatcgaat actcctagag aaaagggaag tcgatctctg 15660agtattgaaa tcgaagtgca catttttttt caacgtgtcc aatcaatcca caaacaaagc 15720agaagacagg taatctttca tacttatact gacaagtaat agtcttaccg tcatgcataa 15780taacgtctcg ttccttcaag aggggttttc cgacatccat aacgacccga agcctcatga 15840aagcattagg gaagaacttt tggttcttct tgtcatggcc tttataggtg tcagccgagc 15900tcgccaattc ccgtccgact ggctccgcaa aatattcgaa cggcaagtta tggacttgca 15960accataactc cacggtattg agcaggacct attgtgaaga ctcatctcat ggagcttcag 16020aatgtggttg tcagcaaacc aatgaccgaa atccatcaca tgacggacgt ccagtgggtg 16080agcgaaacga aacaggaagc gcctatcttt cagagtcgtg agctccacac cggattccgg 16140caactacgtg ttgggcaggc ttcgccgtat tagagatatg ttgaggcaga cccatctgtg 16200ccactcgtac aattacgaga gttgtttttt ttgtgatttt cctagtttct cgttgatggt 16260gagctcatat tctacatcgt atggtctctc aacgtcgttt cctgtcatct gatatcccgt 16320catttgcatc cacgtgcgcc gcctcccgtg ccaagtccct aggtgtcatg cacgccaaat 16380tggtggtggt gcgggctgcc ctgtgcttct taccgatggg tggaggttga gtttgggggt 16440ctccgcggcg atggtagtgg gttgacggtt tggtgtgggt tgacggcatt gatcaattta 16500cttcttgctt caaattcttt ggcagaaaac aattcattag attagaactg gaaaccagag 16560tgatgagacg gattaagtca gattccaaca gagttacatc tcttaagaaa taatgtaacc

16620cctttagact ttatatattt gcaattaaaa aaataattta acttttagac tttatatata 16680gttttaataa ctaagtttaa ccactctatt atttatatcg aaactatttg tatgtctccc 16740ctctaaataa acttggtatt gtgtttacag aacctataat caaataatca atactcaact 16800gaagtttgtg cagttaattg aagggattaa cggccaaaat gcactagtat tatcaaccga 16860atagattcac actagatggc catttccatc aatatcatcg ccgttcttct tctgtccaca 16920tatcccctct gaaacttgag agacacctgc acttcattgt ccttattacg tgttacaaaa 16980tgaaacccat gcatccatgc aaactgaaga atggcgcaag aacccttccc ctccatttct 17040tatgtggcga ccatccattt caccatctcc cgctataaaa cacccccatc acttcaccta 17100gaacatcatc actacttgct tatccatcca aaagataccc acttttacaa caattaccaa 17160caacaacaaa caacaaacaa cattacaatt acatttacaa ttaccatacc atgccaccta 17220gcgctgctaa gcaaatggga gcttctactg gtgttcatgc tggtgttact gactcttctg 17280ctttcaccag aaaggatgtt gctgatagac ctgatctcac catcgttgga gattctgttt 17340acgatgctaa ggctttcaga tctgagcatc ctggtggtgc tcatttcgtt tctttgttcg 17400gaggaagaga tgctactgag gctttcatgg aataccatag aagggcttgg cctaagtcta 17460gaatgtctag attccacgtt ggatctcttg cttctactga ggaacctgtt gctgctgatg 17520agggatacct tcaactttgt gctaggatcg ctaagatggt gccttctgtt tcttctggat 17580tcgctcctgc ttcttactgg gttaaggctg gacttatcct tggatctgct atcgctcttg 17640aggcttacat gctttacgct ggaaagagac ttctcccttc tatcgttctt ggatggcttt 17700tcgctcttat cggtcttaac atccagcatg atgctaacca tggtgctttg tctaagtctg 17760cttctgttaa ccttgctctt ggactttgtc aggattggat cggaggatct atgatccttt 17820ggcttcaaga gcatgttgtt atgcaccacc tccacactaa cgatgttgat aaggatcctg 17880atcaaaaggc tcacggtgct cttagactca agcctactga tgcttggtca cctatgcatt 17940ggcttcagca tctttacctt ttgcctggtg agactatgta cgctttcaag cttttgttcc 18000tcgacatctc tgagcttgtt atgtggcgtt gggagggtga gcctatctct aagcttgctg 18060gatacctctt tatgccttct ttgcttctca agcttacctt ctgggctaga ttcgttgctt 18120tgcctcttta ccttgctcct tctgttcata ctgctgtgtg tatcgctgct actgttatga 18180ctggatcttt ctacctcgct ttcttcttct tcatctccca caacttcgag ggtgttgctt 18240ctgttggacc tgatggatct atcacttcta tgactagagg tgctagcttc cttaagagac 18300aagctgagac ttcttctaac gttggaggac ctcttcttgc tactcttaac ggtggactca 18360actaccaaat tgagcatcac ttgttcccta gagttcacca tggattctac cctagacttg 18420ctcctcttgt taaggctgag cttgaggcta gaggaatcga gtacaagcac taccctacta 18480tctggtctaa ccttgcttct accctcagac atatgtacgc tcttggaaga aggcctagat 18540ctaaggctga gtaatgacaa gcttatgtga cgtgaaataa taacggtaaa atatatgtaa 18600taataataat aataaagcca caaagtgaga atgaggggaa ggggaaatgt gtaatgagcc 18660agtagccggt ggtgctaatt ttgtatcgta ttgtcaataa atcatgaatt ttgtggtttt 18720tatgtgtttt tttaaatcat gaattttaaa ttttataaaa taatctccaa tcggaagaac 18780aacattccat atccatgcat ggatgtttct ttacccaaat ctagttcttg agaggatgaa 18840gcatcaccga acagttctgc aactatccct caaaagcttt aaaatgaaca acaaggaaca 18900gagcaacgtt ccaaagatcc caaacgaaac atattatcta tactaatact atattattaa 18960ttactactgc ccggaatcac aatccctgaa tgattcctat taactacaag ccttgttggc 19020ggcggagaag tgatcggcgc ggcgagaagc agcggactcg gagacgaggc cttggaagat 19080ctgagtcgaa cgggcagaat cagtattttc cttcgacgtt aattgatcct acactatgta 19140ggtcatatcc atcgttttaa tttttggcca ccattcaatt ctgtcttgcc tttagggatg 19200tgaatatgaa cggccaaggt aagagaataa aaataatcca aattaaagca agagaggcca 19260agtaagataa tccaaatgta cacttgtcat tgccaaaatt agtaaaatac tcggcatatt 19320gtattcccac acattattaa aataccgtat atgtattggc tgcatttgca tgaataatac 19380tacgtgtaag cccaaaagaa cccacgtgta gcccatgcaa agttaacact cacgacccca 19440ttcctcagtc tccactatat aaacccacca tccccaatct caccaaaccc accacacaac 19500tcacaactca ctctcacacc ttaaagaacc aatcaccacc aaaaatttta caacaattac 19560caacaacaac aaacaacaaa caacattaca attacattta caattaccat accatgagcg 19620ctgttaccgt tactggatct gatcctaaga acagaggatc ttctagcaac accgagcaag 19680aggttccaaa agttgctatc gataccaacg gaaacgtgtt ctctgttcct gatttcacca 19740tcaaggacat ccttggagct atccctcatg agtgttacga gagaagattg gctacctctc 19800tctactacgt gttcagagat atcttctgca tgcttaccac cggatacctt acccataaga 19860tcctttaccc tctcctcatc tcttacacct ctaacagcat catcaagttc actttctggg 19920ccctttacac ttacgttcaa ggacttttcg gaaccggaat ctgggttctc gctcatgagt 19980gtggacatca agctttctct gattacggaa tcgtgaacga tttcgttgga tggacccttc 20040actcttacct tatggttcct tacttcagct ggaagtactc tcatggaaag caccataagg 20100ctactggaca catgaccaga gatatggttt tcgttcctgc caccaaagag gaattcaaga 20160agtctaggaa cttcttcggt aacctcgctg agtactctga ggattctcca cttagaaccc 20220tttacgagct tcttgttcaa caacttggag gatggatcgc ttacctcttc gttaacgtta 20280caggacaacc ttaccctgat gttccttctt ggaaatggaa ccacttctgg cttacctctc 20340cacttttcga gcaaagagat gctctctaca tcttcctttc tgatcttgga atcctcaccc 20400agggaatcgt tcttactctt tggtacaaga aattcggagg atggtccctt ttcatcaact 20460ggttcgttcc ttacatctgg gttaaccact ggctcgtttt catcacattc cttcagcaca 20520ctgatcctac tatgcctcat tacaacgctg aggaatggac tttcgctaag ggtgctgctg 20580ctactatcga tagaaagttc ggattcatcg gacctcacat cttccatgat atcatcgaga 20640ctcatgtgct tcaccactac tgttctagga tcccattcta caacgctaga cctgcttctg 20700aggctatcaa gaaagttatg ggaaagcact acaggtctag cgacgagaac atgtggaagt 20760cactttggaa gtctttcagg tcttgccaat acgttgacgg tgataacggt gttctcatgt 20820tccgtaacat caacaactgc ggagttggag ctgctgagaa gtaatgaagg ggtgatcgat 20880tatgagatcg tacaaagaca ctgctaggtg ttaaggatgg ataataataa taataatgag 20940atgaatgtgt tttaagttag tgtaacagct gtaataaaga gagagagaga gagagagaga 21000gagagagaga gagagagaga gagagagagg ctgatgaaat gttatgtatg tttcttggtt 21060tttaaaataa atgaaagcac atgctcgtgt ggttctatcg aattattcgg cggttcctgt 21120gggaaaaagt ccagaagggc cgccgcagct actactacaa ccaaggccgt ggaggagggc 21180aacagagcca gcacttcgat agctgctgcg atgatcttaa gcaattgagg agcgagtgca 21240catgcagggg actggagcgt gcaatcggcc agatgaggca ggacatccag cagcagggac 21300agcagcagga agttgagagg tggtcccatc aatctaaaca agtcgctagg gaccttccgg 21360gacagtgcgg cacccagcct agccgatgcc agctccaggg gcagcagcag tctgcatggt 21420tttgaagtgg tgatcgatga gatcgtataa agacactgct aggtgttaag gatgggataa 21480taagatgtgt tttaagtcat taaccgtaat aaaaagagag agaggctgat ggaatgttat 21540gtatgtatgt ttcttggttt ttaaaattaa atggaaagca catgctcgtg tgggttctat 21600ctcgattaaa aatcccaatt atatttggtc taatttagtt tggtattgag taaaacaaat 21660tcgaaccaaa ccaaaatata aatatatagt ttttatatat atgcctttaa gactttttat 21720agaattttct ttaaaaaata tctagaaata tttgcgactc ttctggcatg taatatttcg 21780ttaaatatga agtgctccat ttttattaac tttaaataat tggttgtacg atcactttct 21840tatcaagtgt tactaaaatg cgtcaatctc tttgttcttc catattcata tgtcaaaatc 21900tatcaaaatt cttatatatc tttttcgaat ttgaagtgaa atttcgataa tttaaaatta 21960aatagaacat atcattattt aggtatcata ttgattttta tacttaatta ctaaatttgg 22020ttaactttga aagtgtacat caacgaaaaa ttagtcaaac gactaaaata aataaatatc 22080atgtgttatt aagaaaattc tcctataaga atattttaat agatcatatg tttgtaaaaa 22140aaattaattt ttactaacac atatatttac ttatcaaaaa tttgacaaag taagattaaa 22200ataatattca tctaacaaaa aaaaaaccag aaaatgctga aaacccggca aaaccgaacc 22260aatccaaacc gatatagttg gtttggtttg attttgatat aaaccgaacc aactcggtcc 22320atttgcaccc ctaatcataa tagctttaat atttcaagat attattaagt taacgttgtc 22380aatatcctgg aaattttgca aaatgaatca agcctatatg gctgtaatat gaatttaaaa 22440gcagctcgat gtggtggtaa tatgtaattt acttgattct aaaaaaatat cccaagtatt 22500aataatttct gctaggaaga aggttagcta cgatttacag caaagccaga atacaaagaa 22560ccataaagtg attgaagctc gaaatatacg aaggaacaaa tatttttaaa aaaatacgca 22620atgacttgga acaaaagaaa gtgatatatt ttttgttctt aaacaagcat cccctctaaa 22680gaatggcagt tttcctttgc atgtaactat tatgctccct tcgttacaaa aattttggac 22740tactattggg aacttcttct gaaaatagtc ctgcaggcta gtagattggt tggttggttt 22800ccatgtacca gaaggcttac cctattagtt gaaagttgaa actttgttcc ctactcaatt 22860cctagttgtg taaatgtatg tatatgtaat gtgtataaaa cgtagtactt aaatgactag 22920gagtggttct tgagaccgat gagagatggg agcagaacta aagatgatga cataattaag 22980aacgaatttg aaaggctctt aggtttgaat cctattcgag aatgtttttg tcaaagatag 23040tggcgatttt gaaccaaaga aaacatttaa aaaatcagta tccggttacg ttcatgcaaa 23100tagaaagtgg tctaggatct gattgtaatt ttagacttaa agagtctctt aagattcaat 23160cctggctgtg tacaaaacta caaataatat attttagact atttggcctt aactaaactt 23220ccactcatta tttactgagg ttagagaata gacttgcgaa taaacacatt cccgagaaat 23280actcatgatc ccataattag tcagagggta tgccaatcag atctaagaac acacattccc 23340tcaaatttta atgcacatgt aatcatagtt tagcacaatt caaaaataat gtagtattaa 23400agacagaaat ttgtagactt ttttttggcg ttaaaagaag actaagttta tacgtacatt 23460ttattttaag tggaaaaccg aaattttcca tcgaaatata tgaatttagt atatatattt 23520ctgcaatgta ctattttgct attttggcaa ctttcagtgg actactactt tattacaatg 23580tgtatggatg catgagtttg agtatacaca tgtctaaatg catgctttgt aaaacgtaac 23640ggaccacaaa agaggatcca tacaaataca tctcatagct tcctccatta ttttccgaca 23700caaacagagc attttacaac aattaccaac aacaacaaac aacaaacaac attacaatta 23760catttacaat taccatacca tggaatttgc tcaacctctc gttgctatgg ctcaagagca 23820gtacgctgct atcgatgctg ttgttgctcc tgctatcttc tctgctaccg actctattgg 23880atggggactc aagcctatct cttctgctac taaggatctc cctctcgttg aatctcctac 23940ccctcttatc ctttctctcc tcgcttactt cgctatcgtt ggttctggac tcgtttaccg 24000taaagtgttc cctagaaccg ttaagggaca ggatcctttc cttctcaagg ctcttatgct 24060cgctcacaac gttttcctta tcggactcag cctttacatg tgcctcaagc tcgtttacga 24120ggcttacgtg aacaagtact ccttctgggg aaacgcttac aaccctgctc aaaccgagat 24180ggctaaggtg atctggatct tctacgtgtc caagatctac gagttcatgg acaccttcat 24240catgcttctc aagggaaacg ttaaccaggt ttccttcctc catgtttacc accacggatc 24300tatctctgga atctggtgga tgatcactta tgctgctcca ggtggagatg cttacttctc 24360tgctgctctc aactcttggg ttcatgtgtg catgtacacc tactacttca tggctgctgt 24420tcttcctaag gacgaaaaga ccaagagaaa gtacctttgg tggggaagat accttaccca 24480gatgcaaatg ttccagttct tcatgaacct tctccaggct gtttacctcc tctactcttc 24540ttctccttac cctaagttca ttgctcaact cctcgttgtt tacatggtta ccctcctcat 24600gcttttcgga aacttctact acatgaagca ccacgcttct aagtgataag ggccgccgcc 24660atgtgacaga tcgaaggaag aaagtgtaat aagacgactc tcactactcg atcgctagtg 24720attgtcattg ttatatataa taatgttatc tttcacaact tatcgtaatg catgtgaaac 24780tataacacat taatcctact tgtcatatga taacactctc cccatttaaa actcttgtca 24840atttaaagat ataagattct ttaaatgatt aaaaaaaata tattataaat tcaatcactc 24900ctactaataa attattaatt attatttatt gattaaaaaa atacttatac taatttagtc 24960tgaatagaat aattagattc tagcctgcag ggcggccgcg gatcccatgg agtcaaagat 25020tcaaatagag gacctaacag aactcgccgt aaagactggc gaacagttca tacagagtct 25080cttacgactc aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acacacttgt 25140ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca 25200acaaagggta atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat 25260tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa 25320ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 25380gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 25440tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc 25500tatataagga agttcatttc atttggagag aacacggggg actgaattaa atatgagccc 25560tgagaggcgt cctgttgaaa tcagacctgc tactgctgct gatatggctg ctgtttgtga 25620tatcgtgaac cactacatcg agacttctac cgttaacttc agaactgagc ctcaaactcc 25680tcaagagtgg atcgatgatc ttgagagact ccaagataga tacccttggc ttgttgctga 25740ggttgagggt gttgttgctg gaatcgctta cgctggacct tggaaggcta gaaacgctta 25800cgattggact gttgagtcta ccgtttacgt ttcacacaga catcagagac ttggacttgg 25860atctaccctt tacactcacc ttctcaagtc tatggaagct cagggattca agtctgttgt 25920tgctgttatc ggactcccta acgatccttc tgttagactt catgaggctc ttggatacac 25980tgctagagga actcttagag ctgctggata caagcacggt ggatggcatg atgttggatt 26040ctggcaaaga gatttcgagc ttcctgctcc tcctagacct gttagaccag ttactcagat 26100ctgaatttgc gtgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt 26160gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt 26220aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 26280tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc 26340gcggtgtcat ctatgttact agatcactag tgatgtacgg ttaaaaccac cccagtacat 26400taaaaacgtc cgcaatgtgt tattaagttg tctaagcgtc aatttgttta caccacaata 26460tatcctgcca ccagccagcc aacagctccc cgaccggcag ctcggcacaa aatcaccact 26520cgatacaggc agcccatcag tcc 26543723760DNAArtificial SequencepGA7- mod_G nucleotide sequence 7tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180tcacgacgtt gtaaaacggg cgccccgcgg aaagcttgcg gccgcggtac cgcccgttcg 240actcagatct tccaaggcct cgtctccgag tccgctgctt ctcgccgcgc cgatcacttc 300tccgccgcca acaaggcttg tagttaatag gaatcattca gggattgtga ttccgggcag 360tagtaattaa taatatagta ttagtataga taatatgttt cgtttgggat ctttggaacg 420ttgctctgtt ccttgttgtt cattttaaag cttttgaggg atagttgcag aactgttcgg 480tgatgcttca tcctctcaag aactagattt gggtaaagaa acatccatgc atggatatgg 540aatgttgttc ttccgattgg agattatttt ataaaattta aaattcatga tttaaaaaaa 600cacataaaaa ccacaaaatt catgatttat tgacaatacg atacaaaatt agcaccaccg 660gctactggct cattacacat ttccccttcc cctcattctc actttgtggc tttattatta 720ttattattac atatatttta ccgttattat ttcacgtcac ataagcttgt taattaatta 780tcactgcttc ttagcaccct tagacttgta cctcttacgg tagaagttgg cgaagagaac 840gaacatgttc accataaccc agagttgaag gtaaggaagc cagaaagcgg tattctgaac 900gtaagcggtg tagatagagt gagaagcaca gatacagaac tgaagcatct ggatctgggt 960gatgtacttc ttccagaaac agttaactcc aagagcagca agtccgtagt aagagtacat 1020gatcacgtgc acgaaggtgt taacagaaga tccgaagtag caatctccaa caggctcaag 1080cttcataaca acgaaccatg accagatgag aagagtgtgg tggtagatgt gaaggaaaga 1140aagctggtcg aacttcttcc tcatcaccat gaagaaggtg tcgagaagct caacgtactt 1200gttgttgtag tgaagccaga taacctgaga gattccccaa gagttagcag tcatatcagg 1260gatgtttccc caaaccttaa gtccctgagc cctatgagaa gtaacgaaaa ggtagatgca 1320gtagctgttg aagaaggtct ggtagaagtt gtaaacgagc atagcgttct tgagtccgaa 1380aggttgagat ctgttctgca tgatacgctt tccgaagtag atgaagagga ggtatccaat 1440agttccgata gtaggtcccc agtactcaac ctgcatgaag taagcaggaa ctttctcaga 1500agctgggata tcagggttag ccacattgta ggtaacgtat ccaagagttc cagcaagagc 1560agcagggata gcgatagagg ccatggtatg gtaattgtaa atgtaattgt aatgttgttt 1620gttgtttgtt gttgttggta attgttgtaa aattaattaa gtgggtatct tttggatgga 1680taagcaagta gtgatgatgt tctaggtgaa gtgatggggg tgttttatag cgggagatgg 1740tgaaatggat ggtcgccaca taagaaatgg aggggaaggg ttcttgcgcc attcttcagt 1800ttgcatggat gcatgggttt cattttgtaa cacgtaataa ggacaatgaa gtgcaggtgt 1860ctctcaagtt tcagagggga tatgtggaca gaagaagaac ggcgatgata ttgatggaaa 1920tggccatcta gtgtgaatct attcggttga taatactagt gcattttggc cgttaatccc 1980ttcaattaac tgcacaaact tcagttgagt attgattatt tgattatagg ttctgtaaac 2040acaataccaa gtttatttag aggggagaca tacaaatagt ttcgatataa ataatagagt 2100ggttaaactt agttattaaa actatatata aagtctaaaa gttaaattat ttttttaatt 2160gcaaatatat aaagtctaaa ggggttacat tatttcttaa gagatgtaac tctgttggaa 2220tctgacttaa tccgtctcat cactctggtt tccagttcta atctaatgaa ttgttttctg 2280ccaaagaatt tgaagcaaga agtaaattga tcaatgccgt caacccacac caaaccgtca 2340acccactacc atcgccgcgg agacccccaa actcaacctc cacccatcgg taagaagcac 2400agggcagccc gcaccaccac caatttggcg tgcatgacac ctagggactt ggcacgggag 2460gcggcgcacg tggatgcaaa tgacgggata tcagatgaca ggaaacgacg ttgagagacc 2520atacgatgta gaatatgagc tcaccatcaa cgagaaacta ggaaaatcac aaaaaaaaca 2580actctcgtaa ttgtacgagt ggcacagatg ggtctgcctc aacatatctc taatacggcg 2640aagcctgccc aacacgtagt tgccggaatc cggtgtggag ctcacgactc tgaaagatag 2700gcgcttcctg tttcgtttcg ctcacccact ggacgtccgt catgtgatgg atttcggtca 2760ttggtttgct gacaaccaca ttctgaagct ccatgagatg agtcttcaca ataggtcctg 2820ctcaataccg tggagttatg gttgcaagtc cataacttgc cgttcgaata ttttgcggag 2880ccagtcggac gggaattggc gagctcggct gacacctata aaggccatga caagaagaac 2940caaaagttct tccctaatgc tttcatgagg cttcgggtcg ttatggatgt cggaaaaccc 3000ctcttgaagg aacgagacgt tattatgcat gacggtaaga ctattacttg tcagtataag 3060tatgaaagat tacctgtctt ctgctttgtt tgtggattga ttggacacgt tgaaaaaaaa 3120tgtgcacttc gatttcaata ctcagagatc gacttccctt ttctctagga gtattcgatc 3180aaggcattaa catggaagga agctcaagct ctaaaggctt cacaatggaa cctgaaaaat 3240ttcaacaagc ctaaactgaa atcgaagtca aatcacccaa ccgggagctc taaatcagca 3300aacactcctc ctccacagta tccaatcatc gtgcacgatg ctccaggtat tgcaagccag 3360gtattgcaag ctaggagtag gatagagacc ttaaacgtcg ttggtgtgaa gagtcatctt 3420cagacctaat ggagatagat gtagacggcg gcacgaagac tctgaaacac cagaaaggct 3480agtccaggat aaggatctgc tatcccaact gacctctcgt tagtcccaag gcctctcaac 3540tagagcagga ggaaggatgg tcacaagact aggataatga tgtttccaat atgaacctga 3600atgtccatag ctaatttttt tagtcttgct tctgcacttt ttgtttatta tgttctggtg 3660actatgttat ttacccttgt ccgtatgctt gagggtaccc tagtagattg gttggttggt 3720ttccatgtac cagaaggctt accctattag ttgaaagttg aaactttgtt ccctactcaa 3780ttcctagttg tgtaaatgta tgtatatgta atgtgtataa aacgtagtac ttaaatgact 3840aggagtggtt cttgagaccg atgagagatg ggagcagaac taaagatgat gacataatta 3900agaacgaatt tgaaaggctc ttaggtttga atcctattcg agaatgtttt tgtcaaagat 3960agtggcgatt ttgaaccaaa gaaaacattt aaaaaatcag tatccggtta cgttcatgca 4020aatagaaagt ggtctaggat ctgattgtaa ttttagactt aaagagtctc ttaagattca 4080atcctggctg tgtacaaaac tacaaataat atattttaga ctatttggcc ttaactaaac 4140ttccactcat tatttactga ggttagagaa tagacttgcg aataaacaca ttcccgagaa 4200atactcatga tcccataatt agtcagaggg tatgccaatc agatctaaga acacacattc 4260cctcaaattt taatgcacat gtaatcatag tttagcacaa ttcaaaaata atgtagtatt 4320aaagacagaa atttgtagac ttttttttgg cgttaaaaga agactaagtt tatacgtaca 4380ttttatttta agtggaaaac cgaaattttc catcgaaata tatgaattta gtatatatat 4440ttctgcaatg tactattttg ctattttggc aactttcagt ggactactac tttattacaa 4500tgtgtatgga tgcatgagtt tgagtataca catgtctaaa tgcatgcttt gtaaaacgta 4560acggaccaca aaagaggatc catacaaata catctcatag cttcctccat tattttccga 4620cacaaacaga gcattttaca acaattacca acaacaacaa acaacaaaca acattacaat 4680tacatttaca attaccatac catggaattc gcccagcctc ttgttgctat ggctcaagag 4740caatacgctg ctatcgatgc tgttgttgct cctgctatct tctctgctac tgattctatc 4800ggatggggac ttaagcctat ctcttctgct actaaggact tgcctcttgt tgagtctcct 4860acacctctca tcctttcttt gcttgcttac ttcgctatcg ttggatctgg actcgtttac 4920agaaaggttt tccctagaac cgtgaaggga caagatccat tccttttgaa ggctcttatg 4980cttgctcaca acgtgttcct tatcggactt tctctttaca tgtgcctcaa gcttgtgtac

5040gaggcttacg ttaacaagta ctctttctgg ggaaacgctt acaaccctgc tcaaactgag 5100atggctaagg ttatctggat cttctacgtg agcaagatct acgagttcat ggataccttc 5160atcatgctcc tcaagggaaa tgttaaccag gttagcttcc ttcacgttta ccatcacgga 5220tctatctctg gaatctggtg gatgattact tacgctgctc ctggtggtga tgcttacttc 5280tctgctgctc ttaactcttg ggttcacgtg tgtatgtaca cctactattt tatggctgcc 5340gtgcttccta aggacgagaa aactaagaga aagtacctct ggtggggaag ataccttact 5400caaatgcaga tgttccagtt cttcatgaac cttctccagg ctgtttacct tctctactct 5460tcatctcctt accctaagtt tatcgctcag ctcctcgtgg tgtacatggt tactcttctc 5520atgcttttcg gaaacttcta ctacatgaag caccacgcta gcaagtgatg aggcgcgccg 5580ggccgccgcc atgtgacaga tcgaaggaag aaagtgtaat aagacgactc tcactactcg 5640atcgctagtg attgtcattg ttatatataa taatgttatc tttcacaact tatcgtaatg 5700catgtgaaac tataacacat taatcctact tgtcatatga taacactctc cccatttaaa 5760actcttgtca atttaaagat ataagattct ttaaatgatt aaaaaaaata tattataaat 5820tcaatcactc ctactaataa attattaatt attatttatt gattaaaaaa atacttatac 5880taatttagtc tgaatagaat aattagattc tagtctcatc cccttttaaa ccaacttagt 5940aaacgttttt ttttttaatt ttatgaagtt aagtttttac cttgttttta aaaagaatcg 6000ttcataagat gccatgccag aacattagct acacgttaca catagcatgc agccgcggag 6060aattgttttt cttcgccact tgtcactccc ttcaaacacc taagagcttc tctctcacag 6120cacacacata caatcacatg cgtgcatgca ttattacacg tgatcgccat gcaaatctcc 6180tttatagcct ataaattaac tcatccgctt cactctttac tcaaaccaaa actcatcgat 6240acaaacaaga ttaaaaacat acacgaggat cttttacaac aattaccaac aacaacaaac 6300aacaaacaac attacaatta catttacaat taccatacca tgcctccaag ggactcttac 6360tcttatgctg ctcctccttc tgctcaactt cacgaagttg atactcctca agagcacgac 6420aagaaagagc ttgttatcgg agatagggct tacgatgtta ccaacttcgt taagagacac 6480cctggtggaa agatcattgc ttaccaagtt ggaactgatg ctaccgatgc ttacaagcag 6540ttccatgtta gatctgctaa ggctgacaag atgcttaagt ctcttccttc tcgtcctgtt 6600cacaagggat actctccaag aagggctgat cttatcgctg atttccaaga gttcaccaag 6660caacttgagg ctgagggaat gttcgagcct tctcttcctc atgttgctta cagacttgct 6720gaggttatcg ctatgcatgt tgctggtgct gctcttatct ggcatggata cactttcgct 6780ggaatcgcta tgcttggagt tgttcaggga agatgtggat ggcttatgca tgagggtgga 6840cattactctc tcactggaaa cattgctttc gacagagcta tccaagttgc ttgttacgga 6900cttggatgtg gaatgtctgg tgcttggtgg cgtaaccagc ataacaagca ccatgctact 6960cctcaaaagc ttcagcacga tgttgatctt gatacccttc ctctcgttgc tttccatgag 7020agaatcgctg ctaaggttaa gtctcctgct atgaaggctt ggctttctat gcaagctaag 7080cttttcgctc ctgttaccac tcttcttgtt gctcttggat ggcagcttta ccttcatcct 7140agacacatgc tcaggactaa gcactacgat gagcttgcta tgctcggaat cagatacgga 7200cttgttggat accttgctgc taactacggt gctggatacg ttctcgcttg ttaccttctt 7260tacgttcagc ttggagctat gtacatcttc tgcaacttcg ctgtttctca tactcacctc 7320cctgttgttg agcctaacga gcatgctact tgggttgagt acgctgctaa ccacactact 7380aactgttctc catcttggtg gtgtgattgg tggatgtctt accttaacta ccagatcgag 7440caccaccttt acccttctat gcctcaattc agacacccta agatcgctcc tagagttaag 7500cagcttttcg agaagcacgg acttcactac gatgttagag gatacttcga ggctatggct 7560gatactttcg ctaaccttga taacgttgcc catgctcctg agaagaaaat gcagtaatga 7620gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 7680atgattatca tataatttct gttgaattac gttaagcacg taataattaa catgtaatgc 7740atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 7800gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 7860atgttactag atcggtcgat taaaaatccc aattatattt ggtctaattt agtttggtat 7920tgagtaaaac aaattcgaac caaaccaaaa tataaatata tagtttttat atatatgcct 7980ttaagacttt ttatagaatt ttctttaaaa aatatctaga aatatttgcg actcttctgg 8040catgtaatat ttcgttaaat atgaagtgct ccatttttat taactttaaa taattggttg 8100tacgatcact ttcttatcaa gtgttactaa aatgcgtcaa tctctttgtt cttccatatt 8160catatgtcaa aatctatcaa aattcttata tatctttttc gaatttgaag tgaaatttcg 8220ataatttaaa attaaataga acatatcatt atttaggtat catattgatt tttatactta 8280attactaaat ttggttaact ttgaaagtgt acatcaacga aaaattagtc aaacgactaa 8340aataaataaa tatcatgtgt tattaagaaa attctcctat aagaatattt taatagatca 8400tatgtttgta aaaaaaatta atttttacta acacatatat ttacttatca aaaatttgac 8460aaagtaagat taaaataata ttcatctaac aaaaaaaaaa ccagaaaatg ctgaaaaccc 8520ggcaaaaccg aaccaatcca aaccgatata gttggtttgg tttgattttg atataaaccg 8580aaccaactcg gtccatttgc acccctaatc ataatagctt taatatttca agatattatt 8640aagttaacgt tgtcaatatc ctggaaattt tgcaaaatga atcaagccta tatggctgta 8700atatgaattt aaaagcagct cgatgtggtg gtaatatgta atttacttga ttctaaaaaa 8760atatcccaag tattaataat ttctgctagg aagaaggtta gctacgattt acagcaaagc 8820cagaatacaa agaaccataa agtgattgaa gctcgaaata tacgaaggaa caaatatttt 8880taaaaaaata cgcaatgact tggaacaaaa gaaagtgata tattttttgt tcttaaacaa 8940gcatcccctc taaagaatgg cagttttcct ttgcatgtaa ctattatgct cccttcgtta 9000caaaaatttt ggactactat tgggaacttc ttctgaaaat agtgatagaa cccacacgag 9060catgtgcttt ccatttaatt ttaaaaacca agaaacatac atacataaca ttccatcagc 9120ctctctctct ttttattacg gttaatgact taaaacacat cttattatcc catccttaac 9180acctagcagt gtctttatac gatctcatcg atcaccactt caaaaccatg cagactgctg 9240ctgcccctgg agctggcatc ggctaggctg ggtgccgcac tgtcccggaa ggtccctagc 9300gacttgttta gattgatggg accacctctc aacttcctgc tgctgtccct gctgctggat 9360gtcctgcctc atctggccga ttgcacgctc cagtcccctg catgtgcact cgctcctcaa 9420ttgcttaaga tcatcgcagc agctatcgaa gtgctggctc tgttgccctc ctccacggcc 9480ttggttgtag tagtagctgc cgccgccctt ctggactttt tcccacagga accgccgaat 9540aattcgatag aaccacacga gcatgtgctt tcatttattt taaaaaccaa gaaacataca 9600taacatttca tcagcctctc tctctctctc tctctctctc tctctctctc tctctctctc 9660tctctcttta ttacagctgt tacactaact taaaacacat tcatctcatt attattatta 9720ttatccatcc ttaacaccta gcagtgtctt tgtacgatct cataatcgat caccccttca 9780tcaggtatcc ttaggcttca ctccaacgtt gttgcagtta cggaacatgt acacaccatc 9840atggttctca acgaactggc aagatctcca agttttccaa aggctaaccc acatgttctc 9900atcggtgtgt ctgtagtgct ctcccataac tttcttgatg cactcggtag cttctctagc 9960atggtagaat gggatccttg aaacgtagtg atggagcaca tgagtctcga tgatgtcatg 10020gaagatgatt ccgaggattc cgaactctct atcgatagta gcagcagcac ccttagcgaa 10080agtccactct tgagcatcgt aatgaggcat agaagaatcg gtgtgctgaa ggaaggtaac 10140gaaaacaagc cagtggttaa caaggatcca aggacagaac catgtgatga aagtaggcca 10200gaatccgaaa accttgtaag cggtgtaaac agaagtgagg gtagcaagga ttccaagatc 10260agaaagaacg atgtaccagt agtccttctt atcgaaaaca gggctagaag gccagtagtg 10320agacttgaag aacttagaaa caccagggta aggttgtcca gtagcgttag tagcaaggta 10380aagagaaagt cctccaagct gttggaacaa gagagcgaaa acagagtaga taggagtttc 10440ctcagcgata tcgtgaaggc tggtaacttg gtgcttctct ttgaattcct cggcggtgta 10500aggaacgaaa accatatctc tggtcatgtg tccagtagcc ttatggtgct tagcatgaga 10560gaacttccag ctgaagtaag gaaccataac aagagagtgg agaacccatc caacggtatc 10620gttaacccat ccgtagttag agaaagcaga atgtccacac tcatgtccaa ggatccagat 10680tccgaatccg aaacaagaga tagagaacac gtaagcagac caagcagcga atctaaggaa 10740ttcgttaggg agaagaggga tgtaggtaag tccaacgtaa gcgatagcag agatagccac 10800gatatctctc accacgtaag acatagactt cacgagagat ctctcgtaac agtgcttagg 10860gatagcgtca aggatatcct tgatggtgta atctggcacc ttgaaaacgt ttccgaaggt 10920atcgatagcg gtcttttgct gcttgaaaga tgcaacgttt ccagaacgcc taacggtctt 10980agtagatccc tcaaggatct cagatccaga cacggtaacc ttagacatgg tatggtaatt 11040gtaaatgtaa ttgtaatgtt gtttgttgtt tgttgttgtt ggtaattgtt gtaaaatttt 11100tggtggtgat tggttcttta aggtgtgaga gtgagttgtg agttgtgtgg tgggtttggt 11160gagattgggg atggtgggtt tatatagtgg agactgagga atggggtcgt gagtgttaac 11220tttgcatggg ctacacgtgg gttcttttgg gcttacacgt agtattattc atgcaaatgc 11280agccaataca tatacggtat tttaataatg tgtgggaata caatatgccg agtattttac 11340taattttggc aatgacaagt gtacatttgg attatcttac ttggcctctc ttgctttaat 11400ttggattatt tttattctct taccttggcc gttcatattc acatccctaa aggcaagaca 11460gaattgaatg gtggccaaaa attaaaacga tggatatgac ctacatagtg taggatcaat 11520taacgtcgaa ggaaaatact gattctctca agcatacgga caagggtaaa taacatagtc 11580accagaacat aataaacaaa aagtgcagaa gcaagactaa aaaaattagc tatggacatt 11640caggttcata ttggaaacat cattatccta gtcttgtgac catccttcct cctgctctag 11700ttgagaggcc ttgggactaa cgagaggtca gttgggatag cagatcctta tcctggacta 11760gcctttctgg tgtttcagag tcttcgtgcc gccgtctaca tctatctcca ttaggtctga 11820agatgactct tcacaccaac gacgtttaag gtctctatcc tactcctagc ttgcaatacc 11880tggcttgcaa tacctggagc atcgtgcacg atgattggat actgtggagg aggagtgttt 11940gctgatttag agctcccggt tgggtgattt gacttcgatt tcagtttagg cttgttgaaa 12000tttttcaggt tccattgtga agcctttaga gcttgagctt ccttccatgt taatgccttg 12060atcgaatact cctagagaaa agggaagtcg atctctgagt attgaaatcg aagtgcacat 12120tttttttcaa cgtgtccaat caatccacaa acaaagcaga agacaggtaa tctttcatac 12180ttatactgac aagtaatagt cttaccgtca tgcataataa cgtctcgttc cttcaagagg 12240ggttttccga catccataac gacccgaagc ctcatgaaag cattagggaa gaacttttgg 12300ttcttcttgt catggccttt ataggtgtca gccgagctcg ccaattcccg tccgactggc 12360tccgcaaaat attcgaacgg caagttatgg acttgcaacc ataactccac ggtattgagc 12420aggacctatt gtgaagactc atctcatgga gcttcagaat gtggttgtca gcaaaccaat 12480gaccgaaatc catcacatga cggacgtcca gtgggtgagc gaaacgaaac aggaagcgcc 12540tatctttcag agtcgtgagc tccacaccgg attccggcaa ctacgtgttg ggcaggcttc 12600gccgtattag agatatgttg aggcagaccc atctgtgcca ctcgtacaat tacgagagtt 12660gttttttttg tgattttcct agtttctcgt tgatggtgag ctcatattct acatcgtatg 12720gtctctcaac gtcgtttcct gtcatctgat atcccgtcat ttgcatccac gtgcgccgcc 12780tcccgtgcca agtccctagg tgtcatgcac gccaaattgg tggtggtgcg ggctgccctg 12840tgcttcttac cgatgggtgg aggttgagtt tgggggtctc cgcggcgatg gtagtgggtt 12900gacggtttgg tgtgggttga cggcattgat caatttactt cttgcttcaa attctttggc 12960agaaaacaat tcattagatt agaactggaa accagagtga tgagacggat taagtcagat 13020tccaacagag ttacatctct taagaaataa tgtaacccct ttagacttta tatatttgca 13080attaaaaaaa taatttaact tttagacttt atatatagtt ttaataacta agtttaacca 13140ctctattatt tatatcgaaa ctatttgtat gtctcccctc taaataaact tggtattgtg 13200tttacagaac ctataatcaa ataatcaata ctcaactgaa gtttgtgcag ttaattgaag 13260ggattaacgg ccaaaatgca ctagtattat caaccgaata gattcacact agatggccat 13320ttccatcaat atcatcgccg ttcttcttct gtccacatat cccctctgaa acttgagaga 13380cacctgcact tcattgtcct tattacgtgt tacaaaatga aacccatgca tccatgcaaa 13440ctgaagaatg gcgcaagaac ccttcccctc catttcttat gtggcgacca tccatttcac 13500catctcccgc tataaaacac ccccatcact tcacctagaa catcatcact acttgcttat 13560ccatccaaaa gatacccact tttacaacaa ttaccaacaa caacaaacaa caaacaacat 13620tacaattaca tttacaatta ccataccatg ccacctagcg ctgctaagca aatgggagct 13680tctactggtg ttcatgctgg tgttactgac tcttctgctt tcaccagaaa ggatgttgct 13740gatagacctg atctcaccat cgttggagat tctgtttacg atgctaaggc tttcagatct 13800gagcatcctg gtggtgctca tttcgtttct ttgttcggag gaagagatgc tactgaggct 13860ttcatggaat accatagaag ggcttggcct aagtctagaa tgtctagatt ccacgttgga 13920tctcttgctt ctactgagga acctgttgct gctgatgagg gataccttca actttgtgct 13980aggatcgcta agatggtgcc ttctgtttct tctggattcg ctcctgcttc ttactgggtt 14040aaggctggac ttatccttgg atctgctatc gctcttgagg cttacatgct ttacgctgga 14100aagagacttc tcccttctat cgttcttgga tggcttttcg ctcttatcgg tcttaacatc 14160cagcatgatg ctaaccatgg tgctttgtct aagtctgctt ctgttaacct tgctcttgga 14220ctttgtcagg attggatcgg aggatctatg atcctttggc ttcaagagca tgttgttatg 14280caccacctcc acactaacga tgttgataag gatcctgatc aaaaggctca cggtgctctt 14340agactcaagc ctactgatgc ttggtcacct atgcattggc ttcagcatct ttaccttttg 14400cctggtgaga ctatgtacgc tttcaagctt ttgttcctcg acatctctga gcttgttatg 14460tggcgttggg agggtgagcc tatctctaag cttgctggat acctctttat gccttctttg 14520cttctcaagc ttaccttctg ggctagattc gttgctttgc ctctttacct tgctccttct 14580gttcatactg ctgtgtgtat cgctgctact gttatgactg gatctttcta cctcgctttc 14640ttcttcttca tctcccacaa cttcgagggt gttgcttctg ttggacctga tggatctatc 14700acttctatga ctagaggtgc tagcttcctt aagagacaag ctgagacttc ttctaacgtt 14760ggaggacctc ttcttgctac tcttaacggt ggactcaact accaaattga gcatcacttg 14820ttccctagag ttcaccatgg attctaccct agacttgctc ctcttgttaa ggctgagctt 14880gaggctagag gaatcgagta caagcactac cctactatct ggtctaacct tgcttctacc 14940ctcagacata tgtacgctct tggaagaagg cctagatcta aggctgagta atgacaagct 15000tatgtgacgt gaaataataa cggtaaaata tatgtaataa taataataat aaagccacaa 15060agtgagaatg aggggaaggg gaaatgtgta atgagccagt agccggtggt gctaattttg 15120tatcgtattg tcaataaatc atgaattttg tggtttttat gtgttttttt aaatcatgaa 15180ttttaaattt tataaaataa tctccaatcg gaagaacaac attccatatc catgcatgga 15240tgtttcttta cccaaatcta gttcttgaga ggatgaagca tcaccgaaca gttctgcaac 15300tatccctcaa aagctttaaa atgaacaaca aggaacagag caacgttcca aagatcccaa 15360acgaaacata ttatctatac taatactata ttattaatta ctactgcccg gaatcacaat 15420ccctgaatga ttcctattaa ctacaagcct tgttggcggc ggagaagtga tcggcgcggc 15480gagaagcagc ggactcggag acgaggcctt ggaagatctg agtcgaacgg gcagaatcag 15540tattttcctt cgacgttaat tgatcctaca ctatgtaggt catatccatc gttttaattt 15600ttggccacca ttcaattctg tcttgccttt agggatgtga atatgaacgg ccaaggtaag 15660agaataaaaa taatccaaat taaagcaaga gaggccaagt aagataatcc aaatgtacac 15720ttgtcattgc caaaattagt aaaatactcg gcatattgta ttcccacaca ttattaaaat 15780accgtatatg tattggctgc atttgcatga ataatactac gtgtaagccc aaaagaaccc 15840acgtgtagcc catgcaaagt taacactcac gaccccattc ctcagtctcc actatataaa 15900cccaccatcc ccaatctcac caaacccacc acacaactca caactcactc tcacacctta 15960aagaaccaat caccaccaaa aattttacaa caattaccaa caacaacaaa caacaaacaa 16020cattacaatt acatttacaa ttaccatacc atgagcgctg ttaccgttac tggatctgat 16080cctaagaaca gaggatcttc tagcaacacc gagcaagagg ttccaaaagt tgctatcgat 16140accaacggaa acgtgttctc tgttcctgat ttcaccatca aggacatcct tggagctatc 16200cctcatgagt gttacgagag aagattggct acctctctct actacgtgtt cagagatatc 16260ttctgcatgc ttaccaccgg ataccttacc cataagatcc tttaccctct cctcatctct 16320tacacctcta acagcatcat caagttcact ttctgggccc tttacactta cgttcaagga 16380cttttcggaa ccggaatctg ggttctcgct catgagtgtg gacatcaagc tttctctgat 16440tacggaatcg tgaacgattt cgttggatgg acccttcact cttaccttat ggttccttac 16500ttcagctgga agtactctca tggaaagcac cataaggcta ctggacacat gaccagagat 16560atggttttcg ttcctgccac caaagaggaa ttcaagaagt ctaggaactt cttcggtaac 16620ctcgctgagt actctgagga ttctccactt agaacccttt acgagcttct tgttcaacaa 16680cttggaggat ggatcgctta cctcttcgtt aacgttacag gacaacctta ccctgatgtt 16740ccttcttgga aatggaacca cttctggctt acctctccac ttttcgagca aagagatgct 16800ctctacatct tcctttctga tcttggaatc ctcacccagg gaatcgttct tactctttgg 16860tacaagaaat tcggaggatg gtcccttttc atcaactggt tcgttcctta catctgggtt 16920aaccactggc tcgttttcat cacattcctt cagcacactg atcctactat gcctcattac 16980aacgctgagg aatggacttt cgctaagggt gctgctgcta ctatcgatag aaagttcgga 17040ttcatcggac ctcacatctt ccatgatatc atcgagactc atgtgcttca ccactactgt 17100tctaggatcc cattctacaa cgctagacct gcttctgagg ctatcaagaa agttatggga 17160aagcactaca ggtctagcga cgagaacatg tggaagtcac tttggaagtc tttcaggtct 17220tgccaatacg ttgacggtga taacggtgtt ctcatgttcc gtaacatcaa caactgcgga 17280gttggagctg ctgagaagta atgaaggggt gatcgattat gagatcgtac aaagacactg 17340ctaggtgtta aggatggata ataataataa taatgagatg aatgtgtttt aagttagtgt 17400aacagctgta ataaagagag agagagagag agagagagag agagagagag agagagagag 17460agagaggctg atgaaatgtt atgtatgttt cttggttttt aaaataaatg aaagcacatg 17520ctcgtgtggt tctatcgaat tattcggcgg ttcctgtggg aaaaagtcca gaagggccgc 17580cgcagctact actacaacca aggccgtgga ggagggcaac agagccagca cttcgatagc 17640tgctgcgatg atcttaagca attgaggagc gagtgcacat gcaggggact ggagcgtgca 17700atcggccaga tgaggcagga catccagcag cagggacagc agcaggaagt tgagaggtgg 17760tcccatcaat ctaaacaagt cgctagggac cttccgggac agtgcggcac ccagcctagc 17820cgatgccagc tccaggggca gcagcagtct gcatggtttt gaagtggtga tcgatgagat 17880cgtataaaga cactgctagg tgttaaggat gggataataa gatgtgtttt aagtcattaa 17940ccgtaataaa aagagagaga ggctgatgga atgttatgta tgtatgtttc ttggttttta 18000aaattaaatg gaaagcacat gctcgtgtgg gttctatctc gattaaaaat cccaattata 18060tttggtctaa tttagtttgg tattgagtaa aacaaattcg aaccaaacca aaatataaat 18120atatagtttt tatatatatg cctttaagac tttttataga attttcttta aaaaatatct 18180agaaatattt gcgactcttc tggcatgtaa tatttcgtta aatatgaagt gctccatttt 18240tattaacttt aaataattgg ttgtacgatc actttcttat caagtgttac taaaatgcgt 18300caatctcttt gttcttccat attcatatgt caaaatctat caaaattctt atatatcttt 18360ttcgaatttg aagtgaaatt tcgataattt aaaattaaat agaacatatc attatttagg 18420tatcatattg atttttatac ttaattacta aatttggtta actttgaaag tgtacatcaa 18480cgaaaaatta gtcaaacgac taaaataaat aaatatcatg tgttattaag aaaattctcc 18540tataagaata ttttaataga tcatatgttt gtaaaaaaaa ttaattttta ctaacacata 18600tatttactta tcaaaaattt gacaaagtaa gattaaaata atattcatct aacaaaaaaa 18660aaaccagaaa atgctgaaaa cccggcaaaa ccgaaccaat ccaaaccgat atagttggtt 18720tggtttgatt ttgatataaa ccgaaccaac tcggtccatt tgcaccccta atcataatag 18780ctttaatatt tcaagatatt attaagttaa cgttgtcaat atcctggaaa ttttgcaaaa 18840tgaatcaagc ctatatggct gtaatatgaa tttaaaagca gctcgatgtg gtggtaatat 18900gtaatttact tgattctaaa aaaatatccc aagtattaat aatttctgct aggaagaagg 18960ttagctacga tttacagcaa agccagaata caaagaacca taaagtgatt gaagctcgaa 19020atatacgaag gaacaaatat ttttaaaaaa atacgcaatg acttggaaca aaagaaagtg 19080atatattttt tgttcttaaa caagcatccc ctctaaagaa tggcagtttt cctttgcatg 19140taactattat gctcccttcg ttacaaaaat tttggactac tattgggaac ttcttctgaa 19200aatagtcctg caggctagta gattggttgg ttggtttcca tgtaccagaa ggcttaccct 19260attagttgaa agttgaaact ttgttcccta ctcaattcct agttgtgtaa atgtatgtat 19320atgtaatgcg tataaaacgt agtacttaaa tgactaggag tggttcttga gaccgatgag 19380agatgggagc agaactaaag atgatgacat aattaagaac gaatttgaaa ggctcttagg 19440tttgaatcct attcgagaat gtttttgtca aagatagtgg cgattttgaa ccaaagaaaa 19500catttaaaaa atcagtatcc ggttacgttc atgcaaatag aaagtggtct aggatctgat 19560tgtaatttta gacttaaaga gtctcttaag attcaatcct ggctgtgtac aaaactacaa 19620ataatatatt ttagactatt tggccttaac taaacttcca ctcattattt actgaggtta 19680gagaatagac ttgcgaataa acacattccc gagaaatact catgatccca taattagtca 19740gagggtatgc caatcagatc taagaacaca cattccctca aattttaatg cacatgtaat 19800catagtttag cacaattcaa aaataatgta gtattaaaga cagaaatttg tagacttttt 19860tttggcgtta aaggaagact aagtttatac gtacatttta ttttaagtgg aaaaccgaaa 19920ttttccatcg aaatatatga atttagtata tatatttctg caatgtacta ttttgctatt 19980ttggcaactt tcagtggact actactttat tacaatgtgt atggatgcat gagtttgagt 20040atacacatgt ctaaatgcat gctttgcaaa acgtaacgga ccacaaaaga ggatccatgc

20100aaatacatct catagcttcc tccattattt tccgacacaa acagagcaga ctctagagga 20160tccccccgtt ttacaacaat taccaacaac aacaaacaac aaacaacatt acaattacat 20220ttacaattac catcccaaat cggcgcgcca tgtgtcctcc taagaccgat ggaagatctt 20280ctcctagatc tcctctcacc aggtctaagt catctgctga ggctcttgat gctaaggatg 20340cttctaccgc tcctgttgat cttaagaccc ttgagcctca tgaacttgct gctaccttcg 20400agactagatg ggttagggtt gaggatgttg agtacgacgt gaccaacttc aaacatcctg 20460gtggaagcgt gatcttctac atgcttgcta acactggtgc tgatgctact gaggctttca 20520aagaatttca catgcgtagc ctcaaggctt ggaagatgct tagagctttg ccttctagac 20580ctgctgagat caagagatct gagtctgagg atgctcctat gcttgaggat ttcgctaggt 20640ggagagctga acttgagagg gacggattct tcaagccttc tatcacccat gttgcttacc 20700gtcttttgga gcttcttgct actttcgctc ttggaaccgc tcttatgtac gctggatacc 20760ctatcattgc tagcgttgtg tacggtgctt tcttcggagc tagatgtgga tgggttcaac 20820atgagggtgg acacaactct cttaccggat ctgtgtacgt ggataagaga cttcaggcta 20880tgacttgcgg attcggactt tctaccagcg gagagatgtg gaaccagatg cataacaagc 20940accatgctac ccctcagaaa gttagacacg acatggatct tgataccact cctgctgtgg 21000ctttcttcaa caccgctgtg gaggataata gacctagggg attctctaga gcttgggcta 21060gacttcaagc ttggaccttc gttcctgtta cttctggact tctcgttcag gctttctgga 21120tctacgttct ccatcctaga caggtgctca ggaagaagaa ctacgaggaa gcttcttgga 21180tgctcgtttc tcacgttgtt agaaccgctg ttatcaagct tgctaccgga tactcttggc 21240ctgttgctta ctggtggttc actttcggaa actggatcgc ttacatgtac ctcttcgctc 21300acttctctac ttctcacact cacctccctg ttgttccatc tgacaagcac cttagctggg 21360ttaactacgc tgttgatcac accgttgaca tcgatccttc tcgtggatac gttaactggc 21420ttatgggata ccttaactgc caggttatcc accatctctt ccctgatatg cctcaattca 21480gacagcctga ggtgtcaaga agattcgtcc ctttcgctaa gaagtgggga ctcaactaca 21540aggtgctctc ttactacggt gcttggaagg ctactttcag caacctcgac aaagttggac 21600agcactacta cgttaacgga aaggctgaga aggctcactg atgattaatt aaatttgggc 21660tcgaaccggt tcgagcaagc ttatgtgacg tgaaataata acggtaaaat atatgtaata 21720ataataataa taaagccaca aagtgagaat gaggggaagg ggaaatgtgt aatgagccag 21780tagccggtgg tgctaatttt gtatcgtatt gtcaataaat catgaatttt gtggttttta 21840tgtgtttttt taaatcatga attttaaatt ttataaaata atctccaatc ggaagaacaa 21900cattccatat ccatgcatgg atgtttcttt acccaaatct agttcttgag aggatgaagc 21960atcaccgaac agttctgcaa ctatccctca aaagctttaa aatgaacaac aaggaacaga 22020gcaacgttcc aaagatccca aacgaaacat attatctata ctaatactat attattaatt 22080actactgccc ggaatcacaa tccctgaatg attcctatta actacaagcc ttgttggcgg 22140cggagaagtg atcggcgcgg cgagaagcag cggactcgga gacgaggcct tggaagatct 22200cctgcagggc ggccgcggat cccatggagt caaagattca aatagaggac ctaacagaac 22260tcgccgtaaa gactggcgaa cagttcatac agagtctctt acgactcaat gacaagaaga 22320aaatcttcgt caacatggtg gagcacgaca cacttgtcta ctccaaaaat atcaaagata 22380cagtctcaga agaccaaagg gcaattgaga cttttcaaca aagggtaata tccggaaacc 22440tcctcggatt ccattgccca gctatctgtc actttattgt gaagatagtg gaaaaggaag 22500gtggctccta caaatgccat cattgcgata aaggaaaggc catcgttgaa gatgcctctg 22560ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg 22620ttccaaccac gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg 22680acgcacaatc ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt 22740tggagagaac acgggggact gaattaaata tgagccctga gaggcgtcct gttgaaatca 22800gacctgctac tgctgctgat atggctgctg tttgtgatat cgtgaaccac tacatcgaga 22860cttctaccgt taacttcaga actgagcctc aaactcctca agagtggatc gatgatcttg 22920agagactcca agatagatac ccttggcttg ttgctgaggt tgagggtgtt gttgctggaa 22980tcgcttacgc tggaccttgg aaggctagaa acgcttacga ttggactgtt gagtctaccg 23040tttacgtttc acacagacat cagagacttg gacttggatc taccctttac actcaccttc 23100tcaagtctat ggaagctcag ggattcaagt ctgttgttgc tgttatcgga ctccctaacg 23160atccttctgt tagacttcat gaggctcttg gatacactgc tagaggaact cttagagctg 23220ctggatacaa gcacggtgga tggcatgatg ttggattctg gcaaagagat ttcgagcttc 23280ctgctcctcc tagacctgtt agaccagtta ctcagatctg aatttgcgtg atcgttcaaa 23340catttggcaa taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat 23400ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt 23460tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa 23520caaaatatag cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga 23580tcactagtga tgtacggtta aaaccacccc agtacattaa aaacgtccgc aatgtgttat 23640taagttgtct aagcgtcaat ttgtttacac cacaatatat cctgccacca gccagccaac 23700agctccccga ccggcagctc ggcacaaaat caccactcga tacaggcagc ccatcagtcc 23760811042DNAArtificial SequencepORE04+11ABGBEC_Cowpea_EPA_insert nucleotide sequence 8tcctgtggtt ggcatgcaca tacaaatgga cgaacggata aaccttttca cgccctttta 60aatatccgat tattctaata aacgctcttt tctcttaggt ttacccgcca atatatcctg 120tcaaacactg atagtttaaa ctgaaggcgg gaaacgacaa tctgctagtg gatctcccag 180tcacgacgtt gtaaaacggg cgccctagaa tctaattatt ctattcagac taaattagta 240taagtatttt tttaatcaat aaataataat taataattta ttagtaggag tgattgaatt 300tataatatat tttttttaat catttaaaga atcttatatc tttaaattga caagagtttt 360aaatggggag agtgttatca tatcacaagt aggattaatg tgttatagtt tcacatgcat 420tacgataagt tgtgaaagat aacattatta tatataacaa tgacaatcac tagcgatcga 480gtagtgagag tcgtcttatt acactttctt ccttcgatct gtcacatggc ggcggcccga 540attctcatca cttagaagcg tggtgcttca tgtagtagaa gtttccgaaa agcatgagaa 600gagtcaccat gtacaccacg aggagctgag cgataaactt agggtaaggt gaagaagagt 660agaggaggta cacagcctgg agaaggttca tgaagaactg gaacatttgc atctgggtga 720ggtatcttcc ccaccagagg tactttctct tagttttctc atccttaggg agcacagcag 780ccataaagta gtaggtgtac atgcacacgt gcacccaaga gttgagagca gcagagaaat 840aagcatcacc acctggagca gcgtaggtga tcatccacca gattccagag atagatccgt 900gatggtacac gtggaggaat gacacctggt tcacatttcc cttgaggagc atgatgaagg 960tatccatgaa ctcgtagatc tttgacacgt agaaaatcca gatcacctta gccatctcgg 1020tctgagcagg gttgtaagcg tttccccaga aagagtactt gttcacgtaa gcctcgtaca 1080cgagcttgag gcacatgtag agtgagagtc cgatgaggaa cacgttatga gcgagcatga 1140gagccttgag caagaatgga tcctgtccct tcacagttct agggaacacc tttctgtaca 1200cgagtccaga tcccacgata gcgaagtaag cgaggagaga caagataaga ggggtaggag 1260attccacgag agggagatcc ttagtagcag aagagatagg cttgagtccc catccgatag 1320aatcggtagc agagaagata gcaggagcca caacagcatc gatagcagcg tattgttctt 1380gagccatagc cacgagaggc tgagcaaatt ccatgaattc tgttcttctt tactctttgt 1440gtgactgagg tttggtctag tgctttggtc atctatatat aatgataaca acaatgagaa 1500caagctttgg agtgatcgga gggtctagga tacatgagat tcaagtggac taggatctac 1560accgttggat tttgagtgtg gatatgtgtg aggttaattt tacttggtaa cggccacaaa 1620ggcctaagga gaggtgttga gacccttatc ggcttgaacc gctggaataa tgccacgtgg 1680aagataattc catgaatctt atcgttatct atgagtgaaa ttgtgtgatg gtggagtggt 1740gcttgctcat tttacttgcc tggtggactt ggccctttcc ttatggggaa tttatatttt 1800acttactata gagctttcat accttttttt taccttggat ttagttaata tataatggta 1860tgattcatga ataaaaatgg gaaatttttg aatttgtact gctaaatgca taagattagg 1920tgaaactgtg gaatatatat ttttttcatt taaaagcaaa atttgccttt tactagaatt 1980ataaatatag aaaaatatat aacattcaaa taaaaatgaa aataagaact ttcaaaaaac 2040agaactatgt ttaatgtgta aagattagtc gcacatcaag tcatctgtta caatatgtta 2100caacaagtca taagcccaac aaagttagca cgtctaaata aactaaagag tccacgaaaa 2160tattacaaat cataagccca acaaagttat tgatcaaaaa aaaaaaacgc ccaacaaagc 2220taaacaaagt ccaaaaaaaa cttctcaagt ctccatcttc ctttatgaac attgaaaact 2280atacacaaaa caagtcagat aaatctcttt ctgggcctgt cttcccaacc tcctacatca 2340cttccctatc ggattgaatg ttttacttgt accttttccg ttgcaatgat attgatagta 2400tgtttgtgaa aactaatagg gttaacaatc gaagtcatgg aatatggatt tggtccaaga 2460ttttccgaga gctttctagt agaaagccca tcaccagaaa tttactagta aaataaatca 2520ccaattaggt ttcttattat gtgccaaatt caatataatt atagaggata tttcaaatga 2580aaacgtatga atgttattag taaatggtca ggtaagacat taaaaaaatc ctacgtcaga 2640tattcaactt taaaaattcg atcagtgtgg aattgtacaa aaatttggga tctactatat 2700atatataatg ctttacaaca cttggatttt tttttggagg ctggaatttt taatctacat 2760atttgttttg gccatgcacc aactcattgt ttagtgtaat actttgattt tgtcaaatat 2820atgtgttcgt gtatatttgt ataagaattt ctttgaccat atacacacac acatatatat 2880atatatatat atattatata tcatgcactt ttaattgaaa aaataatata tatatatata 2940gtgcattttt tctaacaacc atatatgttg cgattgatct gcaaaaatac tgctagagta 3000atgaaaaata taatctattg ctgaaattat ctcagatgtt aagattttct taaagtaaat 3060tctttcaaat tttagctaaa agtcttgtaa taactaaaga ataatacaca atctcgacca 3120cggaaaaaaa acacataata aatttggggc ccctagaatc taattattct attcagacta 3180aattagtata agtatttttt taatcaataa ataataatta ataatttatt agtaggagtg 3240attgaattta taatatattt tttttaatca tttaaagaat cttatatctt taaattgaca 3300agagttttaa atggggagag tgttatcata tcacaagtag gattaatgtg ttatagtttc 3360acatgcatta cgataagttg tgaaagataa cattattata tataacaatg acaatcacta 3420gcgatcgagt agtgagagtc gtcttattac actttcttcc ttcgatctgt cacatggcgg 3480cggcccgcgg ccgctcatca gtgagccttc tcagcctttc cgttcacgta gtagtgctgt 3540cccaccttat cgaggtttga gaaggtagcc ttccaagcac cgtagtaaga gagcaccttg 3600tagttgagtc cccacttctt agcgaaagga acgaatcttc ttgacacctc aggctgtctg 3660aactgtggca tatctgggaa gaggtgatgg atcacctggc agttgaggta tcccatgagc 3720cagttcacgt aacccctaga aggatcgata tccacggtgt gatccacagc gtagttcacc 3780caagaaaggt gcttatcaga tggcaccact gggagatggg tatgagaggt agagaagtga 3840gcgaagaggt acatgtaagc gatccagttt ccgaaggtga accaccaata agcaacaggc 3900caagagtatc cggtagcgag cttgataaca gcggttctca caacgtgaga cacgagcatc 3960caagaagcct cttcgtagtt cttctttctg agcacctgtc taggatggag aacgtagatc 4020cagaaagcct gcacgagaag tccagaagtc acaggaacga aagtccaagc ctgaagtcta 4080gcccaagctc tagaaaatcc cctaggcctg ttatcctcaa cagcggtgtt gaagaaagcc 4140acagcaggag tggtatcgag atccatatca tgcctcacct tttgtggggt tgcgtggtgc 4200ttgttgtgca tctggttcca catctcacca gaggtagaaa gtccgaatcc gcaagtcata 4260gcctggagcc tcttatccac atacacagat ccggtgagag agttatgacc accctcgtgt 4320tgaacccatc cacatctagc tccgaagaaa gcaccgtaca ccacagaagc gataataggg 4380tatccagcat acatgagagc agttccgaga gcgaaagtag caagaagctc gagaagtctg 4440tatgccacgt gggtgataga aggcttgaag aatccatccc tctcaagctc agctctccac 4500ctagcgaaat cttcgagcat aggagcatcc tcagactcag acctcttgat ctcagctggt 4560ctagaaggca aagccctaag catcttccaa gccttgagag atctcatgtg aaattctttg 4620aaagcctcag tagcatcagc accggtgtta gcgagcatgt agaagatcac agaaccacca 4680gggtgcttga agttagtaac atcgtactca acatcctcaa ctctcaccca tctagtctcg 4740aaggtagcag ccaactcatg aggctcaaga gtcttgagat ccacaggagc agtagaagca 4800tccttagcat cgagagcctc agcagatgac ttagacctgg taagaggtga cctaggagaa 4860gatcttccat cagtctttgg agggcacatg cggccgctgt tcttctttac tctttgtgtg 4920actgaggttt ggtctagtgc tttggtcatc tatatataat gataacaaca atgagaacaa 4980gctttggagt gatcggaggg tctaggatac atgagattca agtggactag gatctacacc 5040gttggatttt gagtgtggat atgtgtgagg ttaattttac ttggtaacgg ccacaaaggc 5100ctaaggagag gtgttgagac ccttatcggc ttgaaccgct ggaataatgc cacgtggaag 5160ataattccat gaatcttatc gttatctatg agtgaaattg tgtgatggtg gagtggtgct 5220tgctcatttt acttgcctgg tggacttggc cctttcctta tggggaattt atattttact 5280tactatagag ctttcatacc ttttttttac cttggattta gttaatatat aatggtatga 5340ttcatgaata aaaatgggaa atttttgaat ttgtactgct aaatgcataa gattaggtga 5400aactgtggaa tatatatttt tttcatttaa aagcaaaatt tgccttttac tagaattata 5460aatatagaaa aatatataac attcaaataa aaatgaaaat aagaactttc aaaaaacaga 5520actatgttta atgtgtaaag attagtcgca catcaagtca tctgttacaa tatgttacaa 5580caagtcataa gcccaacaaa gttagcacgt ctaaataaac taaagagtcc acgaaaatat 5640tacaaatcat aagcccaaca aagttattga tcaaaaaaaa aaaacgccca acaaagctaa 5700acaaagtcca aaaaaaactt ctcaagtctc catcttcctt tatgaacatt gaaaactata 5760cacaaaacaa gtcagataaa tctctttctg ggcctgtctt cccaacctcc tacatcactt 5820ccctatcgga ttgaatgttt tacttgtacc ttttccgttg caatgatatt gatagtatgt 5880ttgtgaaaac taatagggtt aacaatcgaa gtcatggaat atggatttgg tccaagattt 5940tccgagagct ttctagtaga aagcccatca ccagaaattt actagtaaaa taaatcacca 6000attaggtttc ttattatgtg ccaaattcaa tataattata gaggatattt caaatgaaaa 6060cgtatgaatg ttattagtaa atggtcaggt aagacattaa aaaaatccta cgtcagatat 6120tcaactttaa aaattcgatc agtgtggaat tgtacaaaaa tttgggatct actatatata 6180tataatgctt tacaacactt ggattttttt ttggaggctg gaatttttaa tctacatatt 6240tgttttggcc atgcaccaac tcattgttta gtgtaatact ttgattttgt caaatatatg 6300tgttcgtgta tatttgtata agaatttctt tgaccatata cacacacaca tatatatata 6360tatatatata ttatatatca tgcactttta attgaaaaaa taatatatat atatatagtg 6420cattttttct aacaaccata tatgttgcga ttgatctgca aaaatactgc tagagtaatg 6480aaaaatataa tctattgctg aaattatctc agatgttaag attttcttaa agtaaattct 6540ttcaaatttt agctaaaagt cttgtaataa ctaaagaata atacacaatc tcgaccacgg 6600aaaaaaaaca cataataaat ttgggcgcgc cgcgtattgg ctagagcagc ttgccaacat 6660ggtggagcac gacactctcg tctactccaa gaatatcaaa gatacagtct cagaagacca 6720aagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg gattccattg 6780cccagctatc tgtcacttca tcaaaaggac agtagaaaag gaaggtggca cctacaaatg 6840ccatcattgc gataaaggaa aggctatcgt tcaagatgcc tctgccgaca gtggtcccaa 6900agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc 6960aaagcaagtg gattgatgtg ataacatggt ggagcacgac actctcgtct actccaagaa 7020tatcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac aaagggtaat 7080atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatca aaaggacagt 7140agaaaaggaa ggtggcacct acaaatgcca tcattgcgat aaaggaaagg ctatcgttca 7200agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga 7260aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga 7320cgtaagggat gacgcacaat cccactatcc ttcgcaagac cttcctctat ataaggaagt 7380tcatttcatt tggagaggac acgctgaaat caccagtctc tctctacaaa tctatctctg 7440cgatcgcatg cctcctaggg attcttactc ttacgctgct cctccatctg ctcagctcca 7500tgaagttgat actcctcaag agcacgataa gaaagaactc gtgatcggag atagggctta 7560cgatgtgacc aacttcgtga agagacaccc tggtggaaag attatcgctt accaggttgg 7620aactgatgct accgatgctt acaagcagtt ccacgtgaga tctgctaagg ctgataagat 7680gctcaagtct ctcccatcta ggcctgtgca caagggatat tctccaagaa gggctgatct 7740tatcgctgat ttccaagagt tcaccaagca gcttgaggct gagggaatgt tcgaaccttc 7800tctccctcat gtggcttaca gactcgctga ggttatcgct atgcatgttg ctggtgctgc 7860tctcatctgg cacggatata ctttcgctgg aatcgctatg ctcggagtgg ttcagggaag 7920atgtggatgg cttatgcatg agggtggaca ctactctctc accggaaaca ttgctttcga 7980tagggctatc caggtggcat gctatggact tggatgtgga atgtctggtg cttggtggag 8040aaaccagcat aacaagcacc atgctacccc tcaaaagctc cagcatgatg tggatctcga 8100tactctccct ctcgtggctt tccatgagag aatcgctgct aaggtgaagt ctcctgctat 8160gaaggcttgg ctctctatgc aggctaagct tttcgctcct gtgactactc ttctcgttgc 8220tcttggatgg cagctctacc tccatcctag acacatgctc aggaccaagc actacgatga 8280gcttgctatg ctcggtatca gatacggact cgttggatac ctcgctgcta attacggtgc 8340tggatacgtt ctcgcttgct accttcttta cgttcagctc ggagctatgt acatcttctg 8400caacttcgct gtgtctcaca ctcatctccc tgtggttgaa cctaacgagc atgctacttg 8460ggttgagtac gctgctaacc acactaccaa ctgctctcca tcttggtggt gtgattggtg 8520gatgtcttac ctcaactacc agatcgagca ccacctctac ccttctatgc ctcagttcag 8580acaccctaag atcgctccta gagtgaagca gcttttcgag aagcacggac tccactacga 8640tgtgagagga tactttgagg ctatggctga taccttcgct aacctcgata atgtggctca 8700cgctcctgag aagaaaatgc agtgatgagc gatcgcgatc gttcaaacat ttggcaataa 8760agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 8820aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 8880tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 8940gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcc ctgcagggcg 9000tattggctag agcagcttgc caacatggtg gagcacgaca ctctcgtcta ctccaagaat 9060atcaaagata cagtctcaga agaccaaagg gctattgaga cttttcaaca aagggtaata 9120tcgggaaacc tcctcggatt ccattgccca gctatctgtc acttcatcaa aaggacagta 9180gaaaaggaag gtggcaccta caaatgccat cattgcgata aaggaaaggc tatcgttcaa 9240gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa 9300aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgataa catggtggag 9360cacgacactc tcgtctactc caagaatatc aaagatacag tctcagaaga ccaaagggct 9420attgagactt ttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct 9480atctgtcact tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa atgccatcat 9540tgcgataaag gaaaggctat cgttcaagat gcctctgccg acagtggtcc caaagatgga 9600cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa 9660gtggattgat gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg 9720caagaccttc ctctatataa ggaagttcat ttcatttgga gaggacacgc tgaaatcacc 9780agtctctctc tacaaatcta tctctctcga gatgattgaa caagatggat tgcacgcagg 9840ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 9900ctgctctgat gccgccgtgt tccggctgtc agcgcagggg aggccggttc tttttgtcaa 9960gaccgacctg tccggtgccc tgaatgaact tcaagacgag gcagcgcggc tatcgtggct 10020ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 10080ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 10140cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 10200ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 10260cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 10320gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga ctcatggcga 10380tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 10440ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 10500agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 10560ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaaacg cgtgatcgtt 10620caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta 10680tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt 10740tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag 10800aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac 10860tagatcgacg tccgtacggt taaaaccacc ccagtacatt aaaaacgtcc gcaatgtgtt 10920attaagttgt ctaagcgtca atttgtttac accacaatat atcctgccac cagccagcca 10980acagctcccc gaccggcagc tcggcacaaa atcaccactc gatacaggca gcccatcagt 11040cc 1104291254DNAArtificial SequenceCodon-optimized open reading frame for expression of Lachancea kluyveri 12 desaturase in plants 9atgagcgctg ttaccgttac tggatctgat cctaagaaca gaggatcttc tagcaacacc 60gagcaagagg ttccaaaagt tgctatcgat accaacggaa

acgtgttctc tgttcctgat 120ttcaccatca aggacatcct tggagctatc cctcatgagt gttacgagag aagattggct 180acctctctct actacgtgtt cagagatatc ttctgcatgc ttaccaccgg ataccttacc 240cataagatcc tttaccctct cctcatctct tacacctcta acagcatcat caagttcact 300ttctgggccc tttacactta cgttcaagga cttttcggaa ccggaatctg ggttctcgct 360catgagtgtg gacatcaagc tttctctgat tacggaatcg tgaacgattt cgttggatgg 420acccttcact cttaccttat ggttccttac ttcagctgga agtactctca tggaaagcac 480cataaggcta ctggacacat gaccagagat atggttttcg ttcctgccac caaagaggaa 540ttcaagaagt ctaggaactt cttcggtaac ctcgctgagt actctgagga ttctccactt 600agaacccttt acgagcttct tgttcaacaa cttggaggat ggatcgctta cctcttcgtt 660aacgttacag gacaacctta ccctgatgtt ccttcttgga aatggaacca cttctggctt 720acctctccac ttttcgagca aagagatgct ctctacatct tcctttctga tcttggaatc 780ctcacccagg gaatcgttct tactctttgg tacaagaaat tcggaggatg gtcccttttc 840atcaactggt tcgttcctta catctgggtt aaccactggc tcgttttcat cacattcctt 900cagcacactg atcctactat gcctcattac aacgctgagg aatggacttt cgctaagggt 960gctgctgcta ctatcgatag aaagttcgga ttcatcggac ctcacatctt ccatgatatc 1020atcgagactc atgtgcttca ccactactgt tctaggatcc cattctacaa cgctagacct 1080gcttctgagg ctatcaagaa agttatggga aagcactaca ggtctagcga cgagaacatg 1140tggaagtcac tttggaagtc tttcaggtct tgccaatacg ttgacggtga taacggtgtt 1200ctcatgttcc gtaacatcaa caactgcgga gttggagctg ctgagaagta atga 125410416PRTLachancea kluyveri 10Met Ser Ala Val Thr Val Thr Gly Ser Asp Pro Lys Asn Arg Gly Ser 1 5 10 15 Ser Ser Asn Thr Glu Gln Glu Val Pro Lys Val Ala Ile Asp Thr Asn 20 25 30 Gly Asn Val Phe Ser Val Pro Asp Phe Thr Ile Lys Asp Ile Leu Gly 35 40 45 Ala Ile Pro His Glu Cys Tyr Glu Arg Arg Leu Ala Thr Ser Leu Tyr 50 55 60 Tyr Val Phe Arg Asp Ile Phe Cys Met Leu Thr Thr Gly Tyr Leu Thr 65 70 75 80 His Lys Ile Leu Tyr Pro Leu Leu Ile Ser Tyr Thr Ser Asn Ser Ile 85 90 95 Ile Lys Phe Thr Phe Trp Ala Leu Tyr Thr Tyr Val Gln Gly Leu Phe 100 105 110 Gly Thr Gly Ile Trp Val Leu Ala His Glu Cys Gly His Gln Ala Phe 115 120 125 Ser Asp Tyr Gly Ile Val Asn Asp Phe Val Gly Trp Thr Leu His Ser 130 135 140 Tyr Leu Met Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Gly Lys His 145 150 155 160 His Lys Ala Thr Gly His Met Thr Arg Asp Met Val Phe Val Pro Ala 165 170 175 Thr Lys Glu Glu Phe Lys Lys Ser Arg Asn Phe Phe Gly Asn Leu Ala 180 185 190 Glu Tyr Ser Glu Asp Ser Pro Leu Arg Thr Leu Tyr Glu Leu Leu Val 195 200 205 Gln Gln Leu Gly Gly Trp Ile Ala Tyr Leu Phe Val Asn Val Thr Gly 210 215 220 Gln Pro Tyr Pro Asp Val Pro Ser Trp Lys Trp Asn His Phe Trp Leu 225 230 235 240 Thr Ser Pro Leu Phe Glu Gln Arg Asp Ala Leu Tyr Ile Phe Leu Ser 245 250 255 Asp Leu Gly Ile Leu Thr Gln Gly Ile Val Leu Thr Leu Trp Tyr Lys 260 265 270 Lys Phe Gly Gly Trp Ser Leu Phe Ile Asn Trp Phe Val Pro Tyr Ile 275 280 285 Trp Val Asn His Trp Leu Val Phe Ile Thr Phe Leu Gln His Thr Asp 290 295 300 Pro Thr Met Pro His Tyr Asn Ala Glu Glu Trp Thr Phe Ala Lys Gly 305 310 315 320 Ala Ala Ala Thr Ile Asp Arg Lys Phe Gly Phe Ile Gly Pro His Ile 325 330 335 Phe His Asp Ile Ile Glu Thr His Val Leu His His Tyr Cys Ser Arg 340 345 350 Ile Pro Phe Tyr Asn Ala Arg Pro Ala Ser Glu Ala Ile Lys Lys Val 355 360 365 Met Gly Lys His Tyr Arg Ser Ser Asp Glu Asn Met Trp Lys Ser Leu 370 375 380 Trp Lys Ser Phe Arg Ser Cys Gln Tyr Val Asp Gly Asp Asn Gly Val 385 390 395 400 Leu Met Phe Arg Asn Ile Asn Asn Cys Gly Val Gly Ala Ala Glu Lys 405 410 415 111251DNAPichia pastoris 11atgtctaagg ttaccgtgtc tggatctgag atccttgagg gatctactaa gaccgttagg 60cgttctggaa acgttgcatc tttcaagcag caaaagaccg ctatcgatac cttcggaaac 120gttttcaagg tgccagatta caccatcaag gatatccttg acgctatccc taagcactgt 180tacgagagat ctctcgtgaa gtctatgtct tacgtggtga gagatatcgt ggctatctct 240gctatcgctt acgttggact tacctacatc cctcttctcc ctaacgaatt ccttagattc 300gctgcttggt ctgcttacgt gttctctatc tcttgtttcg gattcggaat ctggatcctt 360ggacatgagt gtggacattc tgctttctct aactacggat gggttaacga taccgttgga 420tgggttctcc actctcttgt tatggttcct tacttcagct ggaagttctc tcatgctaag 480caccataagg ctactggaca catgaccaga gatatggttt tcgttcctta caccgccgag 540gaattcaaag agaagcacca agttaccagc cttcacgata tcgctgagga aactcctatc 600tactctgttt tcgctctctt gttccaacag cttggaggac tttctcttta ccttgctact 660aacgctactg gacaacctta ccctggtgtt tctaagttct tcaagtctca ctactggcct 720tctagccctg ttttcgataa gaaggactac tggtacatcg ttctttctga tcttggaatc 780cttgctaccc tcacttctgt ttacaccgct tacaaggttt tcggattctg gcctactttc 840atcacatggt tctgtccttg gatccttgtt aaccactggc ttgttttcgt taccttcctt 900cagcacaccg attcttctat gcctcattac gatgctcaag agtggacttt cgctaagggt 960gctgctgcta ctatcgatag agagttcgga atcctcggaa tcatcttcca tgacatcatc 1020gagactcatg tgctccatca ctacgtttca aggatcccat tctaccatgc tagagaagct 1080accgagtgca tcaagaaagt tatgggagag cactacagac acaccgatga gaacatgtgg 1140gttagccttt ggaaaacttg gagatcttgc cagttcgttg agaaccatga tggtgtgtac 1200atgttccgta actgcaacaa cgttggagtg aagcctaagg atacctgatg a 125112415PRTPichia pastoris 12Met Ser Lys Val Thr Val Ser Gly Ser Glu Ile Leu Glu Gly Ser Thr 1 5 10 15 Lys Thr Val Arg Arg Ser Gly Asn Val Ala Ser Phe Lys Gln Gln Lys 20 25 30 Thr Ala Ile Asp Thr Phe Gly Asn Val Phe Lys Val Pro Asp Tyr Thr 35 40 45 Ile Lys Asp Ile Leu Asp Ala Ile Pro Lys His Cys Tyr Glu Arg Ser 50 55 60 Leu Val Lys Ser Met Ser Tyr Val Val Arg Asp Ile Val Ala Ile Ser 65 70 75 80 Ala Ile Ala Tyr Val Gly Leu Thr Tyr Ile Pro Leu Leu Pro Asn Glu 85 90 95 Phe Leu Arg Phe Ala Ala Trp Ser Ala Tyr Val Phe Ser Ile Ser Cys 100 105 110 Phe Gly Phe Gly Ile Trp Ile Leu Gly His Glu Cys Gly His Ser Ala 115 120 125 Phe Ser Asn Tyr Gly Trp Val Asn Asp Thr Val Gly Trp Val Leu His 130 135 140 Ser Leu Val Met Val Pro Tyr Phe Ser Trp Lys Phe Ser His Ala Lys 145 150 155 160 His His Lys Ala Thr Gly His Met Thr Arg Asp Met Val Phe Val Pro 165 170 175 Tyr Thr Ala Glu Glu Phe Lys Glu Lys His Gln Val Thr Ser Leu His 180 185 190 Asp Ile Ala Glu Glu Thr Pro Ile Tyr Ser Val Phe Ala Leu Leu Phe 195 200 205 Gln Gln Leu Gly Gly Leu Ser Leu Tyr Leu Ala Thr Asn Ala Thr Gly 210 215 220 Gln Pro Tyr Pro Gly Val Ser Lys Phe Phe Lys Ser His Tyr Trp Pro 225 230 235 240 Ser Ser Pro Val Phe Asp Lys Lys Asp Tyr Trp Tyr Ile Val Leu Ser 245 250 255 Asp Leu Gly Ile Leu Ala Thr Leu Thr Ser Val Tyr Thr Ala Tyr Lys 260 265 270 Val Phe Gly Phe Trp Pro Thr Phe Ile Thr Trp Phe Cys Pro Trp Ile 275 280 285 Leu Val Asn His Trp Leu Val Phe Val Thr Phe Leu Gln His Thr Asp 290 295 300 Ser Ser Met Pro His Tyr Asp Ala Gln Glu Trp Thr Phe Ala Lys Gly 305 310 315 320 Ala Ala Ala Thr Ile Asp Arg Glu Phe Gly Ile Leu Gly Ile Ile Phe 325 330 335 His Asp Ile Ile Glu Thr His Val Leu His His Tyr Val Ser Arg Ile 340 345 350 Pro Phe Tyr His Ala Arg Glu Ala Thr Glu Cys Ile Lys Lys Val Met 355 360 365 Gly Glu His Tyr Arg His Thr Asp Glu Asn Met Trp Val Ser Leu Trp 370 375 380 Lys Thr Trp Arg Ser Cys Gln Phe Val Glu Asn His Asp Gly Val Tyr 385 390 395 400 Met Phe Arg Asn Cys Asn Asn Val Gly Val Lys Pro Lys Asp Thr 405 410 415 131392DNAMicromonas pusilla 13atgtgcccgc cgaagacgga cggccgatcg tccccgcgat cgccgctgac gcgcagcaaa 60tcctccgcgg aggcgctcga cgccaaggac gcgtcgaccg cgcccgtcga tctcaaaacg 120ctcgagccgc acgagctcgc ggcgacgttc gagacgcgat gggtgcgcgt ggaggacgtc 180gagtacgacg tcacaaactt caaacacccg ggaggcagcg tgatattcta catgctcgcg 240aacacgggcg cggacgccac ggaggcgttc aaggagttcc acatgcgatc gcttaaggcg 300tggaagatgc tcagagcgct gccgtcgcgc cccgcggaga tcaaacgcag cgagagcgag 360gacgcgccga tgttggagga tttcgcgcgg tggcgcgcgg agctcgaacg cgacgggttc 420tttaagccct cgataacgca cgtcgcgtat cggttactcg agctcctcgc gaccttcgcc 480ctcggcaccg ccctcatgta cgccgggtac ccgatcatcg cgtccgtcgt gtacggcgcg 540ttcttcggcg ctcggtgcgg ttgggtccag cacgagggcg ggcacaactc gctcacgggg 600tccgtctacg tcgacaagcg cctccaagcg atgacgtgcg ggttcgggct gtccacgagc 660ggggagatgt ggaaccagat gcacaataag caccacgcga cgccgcagaa agtgaggcac 720gacatggacc tggacacgac ccccgcggtg gcgtttttta acaccgccgt ggaggacaac 780cggccgaggg ggttctcccg cgcgtgggct cggcttcagg cgtggacgtt cgtcccggtg 840acctccgggc tgctcgtcca ggcgttctgg atctacgtcc tgcacccgcg gcaggtgttg 900cgaaagaaga actacgagga ggcgtcgtgg atgctcgtct ctcacgtcgt caggaccgcg 960gtgattaaac tcgcgacggg gtactcgtgg cccgtcgcgt actggtggtt caccttcggc 1020aactggatcg cgtacatgta cctcttcgcg cacttctcca cgagccacac gcacctcccg 1080gtcgtgccct cggataagca cctgagctgg gtgaactacg cggtcgatca caccgtggac 1140atcgacccgt cgcgcgggta cgtgaactgg ttgatgggat atctgaactg ccaggtcatt 1200catcacctgt tcccggacat gccgcagttt cgccagccgg aggtgagccg gcggttcgtc 1260ccgttcgcga agaagtgggg gctgaactac aaggtgctgt cctattacgg cgcctggaag 1320gcgacgttct cgaacttgga taaggtcggg cagcactact acgtcaacgg caaggcggag 1380aaggcgcact ga 1392141395DNAArtificial SequenceCodon-optimized open reading frame for expression of Micromonas pusilla 6-desaturase in plants (version 1) 14atgtgccctc ctaagactga tggaagatct tctcctagat ctccacttac caggtctaaa 60tcttctgctg aggctcttga tgctaaggat gcttctactg ctcctgttga tcttaagact 120cttgagcctc atgagcttgc tgctactttc gagactagat gggttagagt tgaggacgtt 180gagtacgatg tgactaactt caagcaccct ggtggatctg tgatcttcta catgcttgct 240aacactggtg ctgatgctac tgaggctttc aaagaattcc acatgcgttc tctcaaggct 300tggaagatgc ttagagcttt gccttctaga cctgctgaga tcaagagatc tgagtctgag 360gatgctccta tgcttgagga tttcgctaga tggcgtgctg agcttgagag agatggattc 420ttcaagcctt ctatcaccca tgtggcttac agacttctcg agcttcttgc tacattcgct 480cttggaactg ctcttatgta cgctggatac cctatcattg cttctgttgt ttacggtgct 540ttcttcggag ctagatgtgg atgggttcaa catgagggtg gacataactc tcttaccgga 600tctgtttacg tggacaagag acttcaggct atgacttgtg gattcggact ttctacttct 660ggtgagatgt ggaaccagat gcataacaag caccatgcta cccctcaaaa ggttagacac 720gatatggatc ttgataccac tcctgctgtg gctttcttca acactgctgt tgaggataac 780agacctagag gattctctag agcttgggct agacttcaag cttggacttt cgttcctgtt 840acctctggac ttcttgttca agctttctgg atctacgttc tccaccctag acaagttctc 900cgtaagaaga actacgaaga ggcttcttgg atgctcgttt ctcatgttgt tagaaccgct 960gttatcaagc ttgctactgg atactcttgg cctgttgctt actggtggtt cactttcgga 1020aactggatcg cttacatgta ccttttcgct cacttctcta cttctcatac tcacctccct 1080gttgttccat ctgataagca cctttcttgg gttaactacg ctgttgatca caccgttgat 1140atcgatcctt ctagaggata cgtgaactgg cttatgggat accttaactg tcaggttatc 1200caccacctct tccctgatat gcctcaattc agacagcctg aggttagcag aagattcgtt 1260cctttcgcta agaagtgggg actcaactac aaggtgctct cttactacgg tgcttggaag 1320gctactttct ctaaccttga taaggtggga cagcactact acgttaacgg aaaggctgag 1380aaggctcact aatga 1395151395DNAArtificial SequenceCodon-optimized open reading frame for expression of Micromonas pusilla 6-desaturase in plants (version 2) 15atgtgtcctc ctaagaccga tggaagatct tctcctagat ctcctctcac caggtctaag 60tcatctgctg aggctcttga tgctaaggat gcttctaccg ctcctgttga tcttaagacc 120cttgagcctc atgaacttgc tgctaccttc gagactagat gggttagggt tgaggatgtt 180gagtacgacg tgaccaactt caaacatcct ggtggaagcg tgatcttcta catgcttgct 240aacactggtg ctgatgctac tgaggctttc aaagaatttc acatgcgtag cctcaaggct 300tggaagatgc ttagagcttt gccttctaga cctgctgaga tcaagagatc tgagtctgag 360gatgctccta tgcttgagga tttcgctagg tggagagctg aacttgagag ggacggattc 420ttcaagcctt ctatcaccca tgttgcttac cgtcttttgg agcttcttgc tactttcgct 480cttggaaccg ctcttatgta cgctggatac cctatcattg ctagcgttgt gtacggtgct 540ttcttcggag ctagatgtgg atgggttcaa catgagggtg gacacaactc tcttaccgga 600tctgtgtacg tggataagag acttcaggct atgacttgcg gattcggact ttctaccagc 660ggagagatgt ggaaccagat gcataacaag caccatgcta cccctcagaa agttagacac 720gacatggatc ttgataccac tcctgctgtg gctttcttca acaccgctgt ggaggataat 780agacctaggg gattctctag agcttgggct agacttcaag cttggacctt cgttcctgtt 840acttctggac ttctcgttca ggctttctgg atctacgttc tccatcctag acaggtgctc 900aggaagaaga actacgagga agcttcttgg atgctcgttt ctcacgttgt tagaaccgct 960gttatcaagc ttgctaccgg atactcttgg cctgttgctt actggtggtt cactttcgga 1020aactggatcg cttacatgta cctcttcgct cacttctcta cttctcacac tcacctccct 1080gttgttccat ctgacaagca ccttagctgg gttaactacg ctgttgatca caccgttgac 1140atcgatcctt ctcgtggata cgttaactgg cttatgggat accttaactg ccaggttatc 1200caccatctct tccctgatat gcctcaattc agacagcctg aggtgtcaag aagattcgtc 1260cctttcgcta agaagtgggg actcaactac aaggtgctct cttactacgg tgcttggaag 1320gctactttca gcaacctcga caaagttgga cagcactact acgttaacgg aaaggctgag 1380aaggctcact gatga 139516463PRTMicromonas pusilla 16Met Cys Pro Pro Lys Thr Asp Gly Arg Ser Ser Pro Arg Ser Pro Leu 1 5 10 15 Thr Arg Ser Lys Ser Ser Ala Glu Ala Leu Asp Ala Lys Asp Ala Ser 20 25 30 Thr Ala Pro Val Asp Leu Lys Thr Leu Glu Pro His Glu Leu Ala Ala 35 40 45 Thr Phe Glu Thr Arg Trp Val Arg Val Glu Asp Val Glu Tyr Asp Val 50 55 60 Thr Asn Phe Lys His Pro Gly Gly Ser Val Ile Phe Tyr Met Leu Ala 65 70 75 80 Asn Thr Gly Ala Asp Ala Thr Glu Ala Phe Lys Glu Phe His Met Arg 85 90 95 Ser Leu Lys Ala Trp Lys Met Leu Arg Ala Leu Pro Ser Arg Pro Ala 100 105 110 Glu Ile Lys Arg Ser Glu Ser Glu Asp Ala Pro Met Leu Glu Asp Phe 115 120 125 Ala Arg Trp Arg Ala Glu Leu Glu Arg Asp Gly Phe Phe Lys Pro Ser 130 135 140 Ile Thr His Val Ala Tyr Arg Leu Leu Glu Leu Leu Ala Thr Phe Ala 145 150 155 160 Leu Gly Thr Ala Leu Met Tyr Ala Gly Tyr Pro Ile Ile Ala Ser Val 165 170 175 Val Tyr Gly Ala Phe Phe Gly Ala Arg Cys Gly Trp Val Gln His Glu 180 185 190 Gly Gly His Asn Ser Leu Thr Gly Ser Val Tyr Val Asp Lys Arg Leu 195 200 205 Gln Ala Met Thr Cys Gly Phe Gly Leu Ser Thr Ser Gly Glu Met Trp 210 215 220 Asn Gln Met His Asn Lys His His Ala Thr Pro Gln Lys Val Arg His 225 230 235 240 Asp Met Asp Leu Asp Thr Thr Pro Ala Val Ala Phe Phe Asn Thr Ala 245 250 255 Val Glu Asp Asn Arg Pro Arg Gly Phe Ser Arg Ala Trp Ala Arg Leu 260 265 270 Gln Ala Trp Thr Phe Val Pro Val Thr Ser Gly Leu Leu Val Gln Ala 275 280 285 Phe Trp Ile Tyr Val Leu His Pro Arg Gln Val Leu Arg Lys Lys Asn 290 295 300 Tyr Glu Glu Ala Ser Trp Met Leu Val Ser His Val Val Arg Thr Ala 305 310 315 320 Val Ile Lys Leu Ala Thr Gly Tyr Ser Trp Pro Val Ala Tyr Trp Trp 325 330 335 Phe Thr Phe Gly Asn Trp Ile Ala Tyr Met Tyr Leu Phe Ala His Phe 340 345 350 Ser Thr Ser His Thr His Leu Pro Val Val Pro Ser Asp Lys His Leu 355 360 365 Ser Trp Val Asn Tyr Ala Val Asp His Thr Val Asp Ile Asp Pro Ser 370 375

380 Arg Gly Tyr Val Asn Trp Leu Met Gly Tyr Leu Asn Cys Gln Val Ile 385 390 395 400 His His Leu Phe Pro Asp Met Pro Gln Phe Arg Gln Pro Glu Val Ser 405 410 415 Arg Arg Phe Val Pro Phe Ala Lys Lys Trp Gly Leu Asn Tyr Lys Val 420 425 430 Leu Ser Tyr Tyr Gly Ala Trp Lys Ala Thr Phe Ser Asn Leu Asp Lys 435 440 445 Val Gly Gln His Tyr Tyr Val Asn Gly Lys Ala Glu Lys Ala His 450 455 460 171449DNAOstreococcus lucimarinus 17atgtgcgtcg aaacgaccga aggcacatcg cgaacgatgg cgaacgaacg cacgagctcg 60tcgtcgtcgc tgagcgaagg cggaacgccg acggtgacgg tcgggatggg aagcgaagac 120gcggggaaga agactcgaaa cgcgagcgtc acggcgtgga cgaaagagtt ggagccgcac 180gcgatcgcga agacgttcga acggcggtac gtgacgatcg aaggcgtgga atacgatgtg 240acggatttta agcatcccgg aggatcggtt atttattaca tgctgtcgaa cacgggagcg 300gacgcgacgg aggcttttaa agagtttcat tatcggtcga aaaaggcgcg caaggcgttg 360gcggcgttgc cgcataagcc agtggacgcg gcgacgcggg aaccgatcga agatgaggcg 420atgctgaagg atttcgcgca gtggcgcaag gaattggagc gtgagggatt ttttaagccc 480tcgccggcgc acgtggcgta tcgattcgcc gagctcgcgg cgatgttcgc gctcggcacg 540gcgttgatgc acgcgcgttg gcacgtcgct tccgtgatcg tgtactcgtg tttcttcggc 600gcgcgatgcg gttgggtgca gcacgagggt gggcacaatt cgttgactgg aaacatttgg 660tgggacaagc gaatccaagc cttcgccgcg gggttcggct tggcgtcgag tggcgacatg 720tggaacaaca tgcacaacaa gcatcacgcg acgccccaaa aggtgcgaca cgatatggat 780ctcgacacca ctcccacggt ggcgttcttc aactccgcgg ttgaagaaaa tcgcccgcgg 840ggattcagta agttgtggtt gcgccttcaa gcgtggacct tcgtgcccgt gacgtccggt 900atggttttgt tcttctggat gttcgtcttg cacccgcgta acgcgctgcg acgcaaaagc 960ttcgaagaag cggcttggat gttttccgcg cacgtcattc gcacggcggt tatcaaagcc 1020gtcaccggct actcctggat cgcctcgtac ggcttgttcg cggcgacgat gtgggcgagc 1080ggatgttact tgttcgcgca cttttccacg tctcacacgc acttggatgt cgtgccgagc 1140gataaacacc tctcgtgggt gcgatacgcc gtcgatcaca cgatcgacat caatccgaac 1200aacagcgtcg tcaactggtt gatgggctac ttgaactgcc aagtcatcca tcacctgttc 1260ccggatatgc ctcagttccg ccaacccgaa gtctcccgcc gattcgtccc gtttgcgaag 1320aagtggaact taaactacaa ggtcttgacg tattatgggg cctggaaggc gacgttcggc 1380aacttgaacg acgtcgggaa gcactattac gtgcacggat ctcagcgcgt caaatcaaag 1440tcggcgtga 1449181449DNAArtificial SequenceCodon-optimized open reading frame for expression of Ostreococcus lucimarinus 6-desaturase in plants 18atgtgtgttg agactactga gggaacctct agaactatgg ctaacgagag gacctcttct 60tcttcttcac tctctgaggg tggaactcct actgttactg tgggaatggg atctgaggat 120gctggaaaga aaaccagaaa cgcttctgtt actgcttgga ccaaagagct tgagcctcac 180gctatcgcta agaccttcga gagaagatac gttaccatcg agggtgttga gtacgatgtg 240accgatttca aacaccctgg tggatctgtg atctactaca tgctctctaa cactggtgct 300gatgctactg aggctttcaa agagttccac taccgttcta agaaggctag aaaggctctt 360gctgctcttc ctcacaagcc tgttgatgct gctactagag agcctattga ggacgaggct 420atgcttaagg atttcgctca gtggagaaaa gagttggaga gagagggatt cttcaagcct 480tctcctgctc atgttgctta ccgtttcgct gaactcgctg ctatgttcgc tcttggaacc 540gctcttatgc atgctagatg gcacgttgct agcgttatcg tgtactcctg tttcttcgga 600gctagatgtg gatgggttca acatgagggt ggacacaact ctcttaccgg aaacatctgg 660tgggataaga gaatccaagc tttcgctgct ggattcggac ttgcttcttc tggtgacatg 720tggaacaaca tgcacaacaa gcaccatgct actcctcaga aagtgagaca cgatatggat 780cttgatacca cccctaccgt tgctttcttc aactctgctg tggaggaaaa cagacctagg 840ggattctcta agctttggct cagacttcaa gcttggacct tcgttcctgt tacctctgga 900atggtgctct tcttctggat gttcgttctc catcctagaa acgctctccg tcgtaagtct 960ttcgaagagg ctgcttggat gttctctgct cacgttatca gaaccgctgt tatcaaggct 1020gttaccggat actcttggat cgctagctac ggacttttcg ctgctactat gtgggcttct 1080ggatgctacc ttttcgctca cttctctact tctcacaccc acctcgatgt tgttccatct 1140gataagcacc ttagctgggt taggtacgct gttgatcaca ccatcgacat caaccctaac 1200aactctgttg tgaactggct tatgggatac cttaactgcc aggttatcca ccatctcttc 1260cctgatatgc ctcaattcag acagcctgag gtgtcaagaa gattcgtccc tttcgctaag 1320aagtggaacc tcaactacaa ggtgctcact tactacggtg cttggaaggc tactttcgga 1380aacctcaacg atgttggaaa gcactactac gttcacggat ctcagagagt gaagagcaag 1440agcgcttga 144919482PRTOstreococcus lucimarinus 19Met Cys Val Glu Thr Thr Glu Gly Thr Ser Arg Thr Met Ala Asn Glu 1 5 10 15 Arg Thr Ser Ser Ser Ser Ser Leu Ser Glu Gly Gly Thr Pro Thr Val 20 25 30 Thr Val Gly Met Gly Ser Glu Asp Ala Gly Lys Lys Thr Arg Asn Ala 35 40 45 Ser Val Thr Ala Trp Thr Lys Glu Leu Glu Pro His Ala Ile Ala Lys 50 55 60 Thr Phe Glu Arg Arg Tyr Val Thr Ile Glu Gly Val Glu Tyr Asp Val 65 70 75 80 Thr Asp Phe Lys His Pro Gly Gly Ser Val Ile Tyr Tyr Met Leu Ser 85 90 95 Asn Thr Gly Ala Asp Ala Thr Glu Ala Phe Lys Glu Phe His Tyr Arg 100 105 110 Ser Lys Lys Ala Arg Lys Ala Leu Ala Ala Leu Pro His Lys Pro Val 115 120 125 Asp Ala Ala Thr Arg Glu Pro Ile Glu Asp Glu Ala Met Leu Lys Asp 130 135 140 Phe Ala Gln Trp Arg Lys Glu Leu Glu Arg Glu Gly Phe Phe Lys Pro 145 150 155 160 Ser Pro Ala His Val Ala Tyr Arg Phe Ala Glu Leu Ala Ala Met Phe 165 170 175 Ala Leu Gly Thr Ala Leu Met His Ala Arg Trp His Val Ala Ser Val 180 185 190 Ile Val Tyr Ser Cys Phe Phe Gly Ala Arg Cys Gly Trp Val Gln His 195 200 205 Glu Gly Gly His Asn Ser Leu Thr Gly Asn Ile Trp Trp Asp Lys Arg 210 215 220 Ile Gln Ala Phe Ala Ala Gly Phe Gly Leu Ala Ser Ser Gly Asp Met 225 230 235 240 Trp Asn Asn Met His Asn Lys His His Ala Thr Pro Gln Lys Val Arg 245 250 255 His Asp Met Asp Leu Asp Thr Thr Pro Thr Val Ala Phe Phe Asn Ser 260 265 270 Ala Val Glu Glu Asn Arg Pro Arg Gly Phe Ser Lys Leu Trp Leu Arg 275 280 285 Leu Gln Ala Trp Thr Phe Val Pro Val Thr Ser Gly Met Val Leu Phe 290 295 300 Phe Trp Met Phe Val Leu His Pro Arg Asn Ala Leu Arg Arg Lys Ser 305 310 315 320 Phe Glu Glu Ala Ala Trp Met Phe Ser Ala His Val Ile Arg Thr Ala 325 330 335 Val Ile Lys Ala Val Thr Gly Tyr Ser Trp Ile Ala Ser Tyr Gly Leu 340 345 350 Phe Ala Ala Thr Met Trp Ala Ser Gly Cys Tyr Leu Phe Ala His Phe 355 360 365 Ser Thr Ser His Thr His Leu Asp Val Val Pro Ser Asp Lys His Leu 370 375 380 Ser Trp Val Arg Tyr Ala Val Asp His Thr Ile Asp Ile Asn Pro Asn 385 390 395 400 Asn Ser Val Val Asn Trp Leu Met Gly Tyr Leu Asn Cys Gln Val Ile 405 410 415 His His Leu Phe Pro Asp Met Pro Gln Phe Arg Gln Pro Glu Val Ser 420 425 430 Arg Arg Phe Val Pro Phe Ala Lys Lys Trp Asn Leu Asn Tyr Lys Val 435 440 445 Leu Thr Tyr Tyr Gly Ala Trp Lys Ala Thr Phe Gly Asn Leu Asn Asp 450 455 460 Val Gly Lys His Tyr Tyr Val His Gly Ser Gln Arg Val Lys Ser Lys 465 470 475 480 Ser Ala 20456PRTOstreococcus lucimarinus 20Met Cys Val Glu Thr Glu Asn Asn Asp Gly Ile Pro Thr Val Glu Ile 1 5 10 15 Ala Phe Asp Gly Glu Arg Glu Arg Ala Glu Ala Asn Val Lys Leu Ser 20 25 30 Ala Glu Lys Met Glu Pro Ala Ala Leu Ala Lys Thr Phe Ala Arg Arg 35 40 45 Tyr Val Val Ile Glu Gly Val Glu Tyr Asp Val Thr Asp Phe Lys His 50 55 60 Pro Gly Gly Thr Val Ile Phe Tyr Ala Leu Ser Asn Thr Gly Ala Asp 65 70 75 80 Ala Thr Glu Ala Phe Lys Glu Phe His His Arg Ser Arg Lys Ala Arg 85 90 95 Lys Ala Leu Ala Ala Leu Pro Ser Arg Pro Ala Lys Thr Ala Lys Val 100 105 110 Asp Asp Ala Glu Met Leu Gln Asp Phe Ala Lys Trp Arg Lys Glu Leu 115 120 125 Glu Arg Asp Gly Phe Phe Lys Pro Ser Pro Ala His Val Ala Tyr Arg 130 135 140 Phe Ala Glu Leu Ala Ala Met Tyr Ala Leu Gly Thr Tyr Leu Met Tyr 145 150 155 160 Ala Arg Tyr Val Val Ser Ser Val Leu Val Tyr Ala Cys Phe Phe Gly 165 170 175 Ala Arg Cys Gly Trp Val Gln His Glu Gly Gly His Ser Ser Leu Thr 180 185 190 Gly Asn Ile Trp Trp Asp Lys Arg Ile Gln Ala Phe Thr Ala Gly Phe 195 200 205 Gly Leu Ala Gly Ser Gly Asp Met Trp Asn Ser Met His Asn Lys His 210 215 220 His Ala Thr Pro Gln Lys Val Arg His Asp Met Asp Leu Asp Thr Thr 225 230 235 240 Pro Ala Val Ala Phe Phe Asn Thr Ala Val Glu Asp Asn Arg Pro Arg 245 250 255 Gly Phe Ser Lys Tyr Trp Leu Arg Leu Gln Ala Trp Thr Phe Ile Pro 260 265 270 Val Thr Ser Gly Leu Val Leu Leu Phe Trp Met Phe Phe Leu His Pro 275 280 285 Ser Lys Ala Leu Lys Gly Gly Lys Tyr Glu Glu Leu Val Trp Met Leu 290 295 300 Ala Ala His Val Ile Arg Thr Trp Thr Ile Lys Ala Val Thr Gly Phe 305 310 315 320 Thr Ala Met Gln Ser Tyr Gly Leu Phe Leu Ala Thr Ser Trp Val Ser 325 330 335 Gly Cys Tyr Leu Phe Ala His Phe Ser Thr Ser His Thr His Leu Asp 340 345 350 Val Val Pro Ala Asp Glu His Leu Ser Trp Val Arg Tyr Ala Val Asp 355 360 365 His Thr Ile Asp Ile Asp Pro Ser Gln Gly Trp Val Asn Trp Leu Met 370 375 380 Gly Tyr Leu Asn Cys Gln Val Ile His His Leu Phe Pro Ser Met Pro 385 390 395 400 Gln Phe Arg Gln Pro Glu Val Ser Arg Arg Phe Val Ala Phe Ala Lys 405 410 415 Lys Trp Asn Leu Asn Tyr Lys Val Met Thr Tyr Ala Gly Ala Trp Lys 420 425 430 Ala Thr Leu Gly Asn Leu Asp Asn Val Gly Lys His Tyr Tyr Val His 435 440 445 Gly Gln His Ser Gly Lys Thr Ala 450 455 21894DNAPyramimonas cordata 21atggagttcg ctcagcctct tgtggctatg gcacaggagc agtatgccgc aattgacgcg 60gtggtagccc ctgcaatttt ctcagctacc gacagcatcg gttggggtct taagcccatt 120agcagcgcga caaaggatct tcctctcgtt gagagtccga cgccgctcat actgagcctg 180ttggcctatt ttgcgatcgt cggctctggg ctggtgtacc gcaaagtatt ccctcgcaca 240gtaaaggggc aagacccctt cctgctgaag gcgctcatgc ttgcgcacaa cgtgttcctc 300attggcctca gtctatacat gtgcttgaag cttgtctacg aggcttacgt caacaagtac 360tccttctggg gaaacgccta caaccccgca cagaccgaga tggcgaaggt catctggatt 420ttctacgtct ccaagatcta tgagttcatg gacacgttca tcatgctctt gaagggcaac 480gtcaaccagg tctctttcct gcatgtgtac catcatggct ccatctctgg tatctggtgg 540atgatcacct acgctgcccc tggcggtgac gcgtacttct cggcggcgct caactcgtgg 600gtgcacgtgt gcatgtacac gtactacttc atggcggcgg tgctgcccaa ggacgagaag 660accaagcgca agtacctctg gtggggccgc tacctgaccc agatgcagat gttccagttc 720ttcatgaacc tgctccaggc ggtctacctc ctctactcct ctagccccta ccccaagttc 780atcgcccagc tgctggtggt gtacatggtc acgctgctga tgctcttcgg caacttctac 840tacatgaagc accacgcgag caagaagcag aagctggcca gcaagaagca gtag 89422870DNAArtificial SequenceCodon-optimized open reading frame for expression of Pyramimonas cordata 6-elongase in plants (truncated at 3' end and encoding functional elongase) (version 1) 22atggaattcg cccagcctct tgttgctatg gctcaagagc aatacgctgc tatcgatgct 60gttgttgctc ctgctatctt ctctgctact gattctatcg gatggggact taagcctatc 120tcttctgcta ctaaggactt gcctcttgtt gagtctccta cacctctcat cctttctttg 180cttgcttact tcgctatcgt tggatctgga ctcgtttaca gaaaggtttt ccctagaacc 240gtgaagggac aagatccatt ccttttgaag gctcttatgc ttgctcacaa cgtgttcctt 300atcggacttt ctctttacat gtgcctcaag cttgtgtacg aggcttacgt taacaagtac 360tctttctggg gaaacgctta caaccctgct caaactgaga tggctaaggt tatctggatc 420ttctacgtga gcaagatcta cgagttcatg gataccttca tcatgctcct caagggaaat 480gttaaccagg ttagcttcct tcacgtttac catcacggat ctatctctgg aatctggtgg 540atgattactt acgctgctcc tggtggtgat gcttacttct ctgctgctct taactcttgg 600gttcacgtgt gtatgtacac ctactatttt atggctgccg tgcttcctaa ggacgagaaa 660actaagagaa agtacctctg gtggggaaga taccttactc aaatgcagat gttccagttc 720ttcatgaacc ttctccaggc tgtttacctt ctctactctt catctcctta ccctaagttt 780atcgctcagc tcctcgtggt gtacatggtt actcttctca tgcttttcgg aaacttctac 840tacatgaagc accacgctag caagtgatga 87023870DNAArtificial SequenceCodon-optimized open reading frame for expression of Pyramimonas cordata 6-elongase in plants (truncated at 3' end and encoding functional elongase) (version 2) 23atggaattcg cccagcctct tgttgctatg gctcaagagc aatacgctgc tatcgatgct 60gttgttgctc ctgctatctt ctctgctact gattctatcg gatggggact taagcctatc 120tcttctgcta ctaaggactt gcctcttgtt gagtctccta cacctctcat cctttctttg 180cttgcttact tcgctatcgt tggatctgga ctcgtttaca gaaaggtttt ccctagaacc 240gtgaagggac aagatccatt ccttttgaag gctcttatgc ttgctcacaa cgtgttcctt 300atcggacttt ctctttacat gtgcctcaag cttgtgtacg aggcttacgt taacaagtac 360tctttctggg gaaacgctta caaccctgct caaactgaga tggctaaggt tatctggatc 420ttctacgtga gcaagatcta cgagttcatg gataccttca tcatgctcct caagggaaat 480gttaaccagg ttagcttcct tcacgtttac catcacggat ctatctctgg aatctggtgg 540atgattactt acgctgctcc tggtggtgat gcttacttct ctgctgctct taactcttgg 600gttcacgtgt gtatgtacac ctactatttt atggctgccg tgcttcctaa ggacgagaaa 660actaagagaa agtacctctg gtggggaaga taccttactc aaatgcagat gttccagttc 720ttcatgaacc ttctccaggc tgtttacctt ctctactctt catctcctta ccctaagttt 780atcgctcagc tcctcgtggt gtacatggtt actcttctca tgcttttcgg aaacttctac 840tacatgaagc accacgctag caagtgatga 87024870DNAArtificial SequenceCodon-optimized open reading frame for expression of Pyramimonas cordata 6-elongase in plants (truncated at 3' end and encoding functional elongase) (version 3) 24atggaatttg ctcaacctct cgttgctatg gctcaagagc agtacgctgc tatcgatgct 60gttgttgctc ctgctatctt ctctgctacc gactctattg gatggggact caagcctatc 120tcttctgcta ctaaggatct ccctctcgtt gaatctccta cccctcttat cctttctctc 180ctcgcttact tcgctatcgt tggttctgga ctcgtttacc gtaaagtgtt ccctagaacc 240gttaagggac aggatccttt ccttctcaag gctcttatgc tcgctcacaa cgttttcctt 300atcggactca gcctttacat gtgcctcaag ctcgtttacg aggcttacgt gaacaagtac 360tccttctggg gaaacgctta caaccctgct caaaccgaga tggctaaggt gatctggatc 420ttctacgtgt ccaagatcta cgagttcatg gacaccttca tcatgcttct caagggaaac 480gttaaccagg tttccttcct ccatgtttac caccacggat ctatctctgg aatctggtgg 540atgatcactt atgctgctcc aggtggagat gcttacttct ctgctgctct caactcttgg 600gttcatgtgt gcatgtacac ctactacttc atggctgctg ttcttcctaa ggacgaaaag 660accaagagaa agtacctttg gtggggaaga taccttaccc agatgcaaat gttccagttc 720ttcatgaacc ttctccaggc tgtttacctc ctctactctt cttctcctta ccctaagttc 780attgctcaac tcctcgttgt ttacatggtt accctcctca tgcttttcgg aaacttctac 840tacatgaagc accacgcttc taagtgataa 87025297PRTPyramimonas cordata 25Met Glu Phe Ala Gln Pro Leu Val Ala Met Ala Gln Glu Gln Tyr Ala 1 5 10 15 Ala Ile Asp Ala Val Val Ala Pro Ala Ile Phe Ser Ala Thr Asp Ser 20 25 30 Ile Gly Trp Gly Leu Lys Pro Ile Ser Ser Ala Thr Lys Asp Leu Pro 35 40 45 Leu Val Glu Ser Pro Thr Pro Leu Ile Leu Ser Leu Leu Ala Tyr Phe 50 55 60 Ala Ile Val Gly Ser Gly Leu Val Tyr Arg Lys Val Phe Pro Arg Thr 65 70 75 80 Val Lys Gly Gln Asp Pro Phe Leu Leu Lys Ala Leu Met Leu Ala His 85 90 95 Asn Val Phe Leu Ile Gly Leu Ser Leu Tyr Met Cys Leu Lys Leu Val 100 105 110 Tyr Glu Ala Tyr Val Asn Lys Tyr Ser Phe Trp Gly Asn Ala Tyr Asn 115 120 125 Pro Ala Gln Thr Glu Met Ala Lys Val Ile Trp Ile Phe Tyr Val Ser 130 135 140 Lys Ile Tyr Glu

Phe Met Asp Thr Phe Ile Met Leu Leu Lys Gly Asn 145 150 155 160 Val Asn Gln Val Ser Phe Leu His Val Tyr His His Gly Ser Ile Ser 165 170 175 Gly Ile Trp Trp Met Ile Thr Tyr Ala Ala Pro Gly Gly Asp Ala Tyr 180 185 190 Phe Ser Ala Ala Leu Asn Ser Trp Val His Val Cys Met Tyr Thr Tyr 195 200 205 Tyr Phe Met Ala Ala Val Leu Pro Lys Asp Glu Lys Thr Lys Arg Lys 210 215 220 Tyr Leu Trp Trp Gly Arg Tyr Leu Thr Gln Met Gln Met Phe Gln Phe 225 230 235 240 Phe Met Asn Leu Leu Gln Ala Val Tyr Leu Leu Tyr Ser Ser Ser Pro 245 250 255 Tyr Pro Lys Phe Ile Ala Gln Leu Leu Val Val Tyr Met Val Thr Leu 260 265 270 Leu Met Leu Phe Gly Asn Phe Tyr Tyr Met Lys His His Ala Ser Lys 275 280 285 Lys Gln Lys Leu Ala Ser Lys Lys Gln 290 295 26288PRTPyramimonas cordata 26Met Glu Phe Ala Gln Pro Leu Val Ala Met Ala Gln Glu Gln Tyr Ala 1 5 10 15 Ala Ile Asp Ala Val Val Ala Pro Ala Ile Phe Ser Ala Thr Asp Ser 20 25 30 Ile Gly Trp Gly Leu Lys Pro Ile Ser Ser Ala Thr Lys Asp Leu Pro 35 40 45 Leu Val Glu Ser Pro Thr Pro Leu Ile Leu Ser Leu Leu Ala Tyr Phe 50 55 60 Ala Ile Val Gly Ser Gly Leu Val Tyr Arg Lys Val Phe Pro Arg Thr 65 70 75 80 Val Lys Gly Gln Asp Pro Phe Leu Leu Lys Ala Leu Met Leu Ala His 85 90 95 Asn Val Phe Leu Ile Gly Leu Ser Leu Tyr Met Cys Leu Lys Leu Val 100 105 110 Tyr Glu Ala Tyr Val Asn Lys Tyr Ser Phe Trp Gly Asn Ala Tyr Asn 115 120 125 Pro Ala Gln Thr Glu Met Ala Lys Val Ile Trp Ile Phe Tyr Val Ser 130 135 140 Lys Ile Tyr Glu Phe Met Asp Thr Phe Ile Met Leu Leu Lys Gly Asn 145 150 155 160 Val Asn Gln Val Ser Phe Leu His Val Tyr His His Gly Ser Ile Ser 165 170 175 Gly Ile Trp Trp Met Ile Thr Tyr Ala Ala Pro Gly Gly Asp Ala Tyr 180 185 190 Phe Ser Ala Ala Leu Asn Ser Trp Val His Val Cys Met Tyr Thr Tyr 195 200 205 Tyr Phe Met Ala Ala Val Leu Pro Lys Asp Glu Lys Thr Lys Arg Lys 210 215 220 Tyr Leu Trp Trp Gly Arg Tyr Leu Thr Gln Met Gln Met Phe Gln Phe 225 230 235 240 Phe Met Asn Leu Leu Gln Ala Val Tyr Leu Leu Tyr Ser Ser Ser Pro 245 250 255 Tyr Pro Lys Phe Ile Ala Gln Leu Leu Val Val Tyr Met Val Thr Leu 260 265 270 Leu Met Leu Phe Gly Asn Phe Tyr Tyr Met Lys His His Ala Ser Lys 275 280 285 271278DNAPavlova salina 27atgccgccgc gcgatagcta ctcgtacgcc gccccgccgt cggcccagct gcacgaggtc 60gataccccgc aggagcatga taagaaggag ctcgtcatcg gtgaccgcgc gtacgacgtg 120accaactttg tgaagcgcca cccgggtggc aagatcatcg cataccaggt tggcacagat 180gcgacggacg cgtacaagca gttccatgtg cggtctgcca aggcggacaa gatgctcaag 240tcgctgcctt cgcgcccggt gcacaagggc tactcgcccc gccgcgctga cctcattgcc 300gacttccagg agttcaccaa gcagctggag gcggagggca tgtttgagcc gtcgctgccg 360cacgtggcat accgcctggc ggaggtgatc gcgatgcacg tggccggcgc cgcgctcatc 420tggcacgggt acaccttcgc gggcattgcc atgctcggcg ttgtgcaggg ccgctgcggc 480tggctcatgc acgagggcgg ccactactcg ctcacgggca acattgcttt tgaccgtgcc 540atccaagtcg cgtgctacgg ccttggctgc ggcatgtcgg gcgcgtggtg gcgcaaccag 600cacaacaagc accacgcgac gccgcagaag ttgcagcacg acgtcgacct cgacaccctc 660ccgctcgtcg ccttccacga gcggatagcc gccaaggtga agagccccgc gatgaaggcg 720tggcttagta tgcaggcgaa gctcttcgcg ccagtgacca cgctgctggt cgcgctgggc 780tggcagctgt acctgcaccc gcgccatatg ctgcgcacca agcactacga cgagctcgcg 840atgctcggca ttcgctacgg ccttgtcggc tacctcgcgg cgaactacgg cgcggggtac 900gtgctcgcgt gctacctgct gtacgtgcag ctcggcgcca tgtacatctt ctgcaacttt 960gccgtgtcgc acacacacct gccggttgtc gagcctaacg agcacgcaac gtgggtggag 1020tacgccgcga accacacgac caactgctcg ccctcgtggt ggtgcgactg gtggatgtcg 1080tacctcaact accagatcga gcaccacctc tacccgtcca tgccgcagtt ccgccacccg 1140aagattgcgc cgcgggtgaa gcagctcttc gagaagcacg gcctgcacta cgacgtgcgt 1200ggctacttcg aggccatggc ggacacgttt gccaaccttg acaacgtcgc gcacgcgccg 1260gagaagaaga tgcagtga 1278281281DNAArtificial SequenceCodon-optimized open reading frame for expression of Pavlova salina 5-desaturase in plants (version 1) 28atgcctccaa gggactctta ctcttatgct gctcctcctt ctgctcaact tcacgaagtt 60gatactcctc aagagcacga caagaaagag cttgttatcg gagatagggc ttacgatgtt 120accaacttcg ttaagagaca ccctggtgga aagatcattg cttaccaagt tggaactgat 180gctaccgatg cttacaagca gttccatgtt agatctgcta aggctgacaa gatgcttaag 240tctcttcctt ctcgtcctgt tcacaaggga tactctccaa gaagggctga tcttatcgct 300gatttccaag agttcaccaa gcaacttgag gctgagggaa tgttcgagcc ttctcttcct 360catgttgctt acagacttgc tgaggttatc gctatgcatg ttgctggtgc tgctcttatc 420tggcatggat acactttcgc tggaatcgct atgcttggag ttgttcaggg aagatgtgga 480tggcttatgc atgagggtgg acattactct ctcactggaa acattgcttt cgacagagct 540atccaagttg cttgttacgg acttggatgt ggaatgtctg gtgcttggtg gcgtaaccag 600cataacaagc accatgctac tcctcaaaag cttcagcacg atgttgatct tgataccctt 660cctctcgttg ctttccatga gagaatcgct gctaaggtta agtctcctgc tatgaaggct 720tggctttcta tgcaagctaa gcttttcgct cctgttacca ctcttcttgt tgctcttgga 780tggcagcttt accttcatcc tagacacatg ctcaggacta agcactacga tgagcttgct 840atgctcggaa tcagatacgg acttgttgga taccttgctg ctaactacgg tgctggatac 900gttctcgctt gttaccttct ttacgttcag cttggagcta tgtacatctt ctgcaacttc 960gctgtttctc atactcacct ccctgttgtt gagcctaacg agcatgctac ttgggttgag 1020tacgctgcta accacactac taactgttct ccatcttggt ggtgtgattg gtggatgtct 1080taccttaact accagatcga gcaccacctt tacccttcta tgcctcaatt cagacaccct 1140aagatcgctc ctagagttaa gcagcttttc gagaagcacg gacttcacta cgatgttaga 1200ggatacttcg aggctatggc tgatactttc gctaaccttg ataacgttgc ccatgctcct 1260gagaagaaaa tgcagtaatg a 1281291281DNAArtificial SequenceCodon-optimized open reading frame for expression of Pavlova salina 5-desaturase in plants (version 2) 29atgcctccta gggactctta ctcttacgct gctcctcctt ctgctcaact tcacgaggtt 60gacactcctc aagagcacga caagaaagag cttgttatcg gagatagggc ttacgatgtg 120accaacttcg ttaagagaca ccctggtgga aagatcattg cttaccaagt gggaactgat 180gctaccgatg cttacaagca gttccatgtg agatctgcta aggctgacaa gatgctcaag 240tctctccctt ctagacctgt tcacaaggga tactctccta gaagagctga tcttatcgct 300gacttccaag agttcactaa gcaacttgag gctgagggaa tgttcgaacc ttctctccct 360catgttgctt accgtcttgc tgaggttatc gctatgcatg ttgctggtgc tgctcttatc 420tggcacggat acactttcgc tggaatcgct atgcttggag ttgttcaggg aagatgcgga 480tggcttatgc atgagggtgg acactactct cttaccggaa acattgcttt cgatagggct 540atccaagttg cttgttacgg acttggatgc ggaatgtctg gtgcttggtg gagaaaccag 600cataacaagc accatgctac tcctcaaaag ctccagcacg atgttgatct tgataccctc 660cctctcgttg ctttccatga gagaatcgct gctaaggtta agtctcctgc tatgaaggct 720tggctctcca tgcaagctaa actcttcgct cctgttacca ctcttcttgt tgctcttgga 780tggcagcttt accttcaccc tagacacatg ctcagaacta agcactacga cgagcttgct 840atgcttggta tcagatacgg acttgtggga taccttgctg ctaactacgg tgctggatac 900gttcttgctt gctaccttct ctacgttcag cttggagcta tgtacatctt ctgcaacttc 960gctgtttctc acactcatct ccctgttgtt gagcctaacg agcatgctac ttgggttgag 1020tacgctgcta accacactac taactgctct ccatcttggt ggtgtgattg gtggatgagc 1080tacctcaact accagatcga gcatcacctt tacccttcta tgcctcagtt caggcatcct 1140aagatcgctc ctagagtgaa gcaactcttc gagaagcacg gacttcacta cgatgtgcgt 1200ggatacttcg aggctatggc tgatactttc gctaacctcg ataacgttgc tcatgctcct 1260gagaagaaaa tgcaatgatg a 128130425PRTPavlova salina 30Met Pro Pro Arg Asp Ser Tyr Ser Tyr Ala Ala Pro Pro Ser Ala Gln 1 5 10 15 Leu His Glu Val Asp Thr Pro Gln Glu His Asp Lys Lys Glu Leu Val 20 25 30 Ile Gly Asp Arg Ala Tyr Asp Val Thr Asn Phe Val Lys Arg His Pro 35 40 45 Gly Gly Lys Ile Ile Ala Tyr Gln Val Gly Thr Asp Ala Thr Asp Ala 50 55 60 Tyr Lys Gln Phe His Val Arg Ser Ala Lys Ala Asp Lys Met Leu Lys 65 70 75 80 Ser Leu Pro Ser Arg Pro Val His Lys Gly Tyr Ser Pro Arg Arg Ala 85 90 95 Asp Leu Ile Ala Asp Phe Gln Glu Phe Thr Lys Gln Leu Glu Ala Glu 100 105 110 Gly Met Phe Glu Pro Ser Leu Pro His Val Ala Tyr Arg Leu Ala Glu 115 120 125 Val Ile Ala Met His Val Ala Gly Ala Ala Leu Ile Trp His Gly Tyr 130 135 140 Thr Phe Ala Gly Ile Ala Met Leu Gly Val Val Gln Gly Arg Cys Gly 145 150 155 160 Trp Leu Met His Glu Gly Gly His Tyr Ser Leu Thr Gly Asn Ile Ala 165 170 175 Phe Asp Arg Ala Ile Gln Val Ala Cys Tyr Gly Leu Gly Cys Gly Met 180 185 190 Ser Gly Ala Trp Trp Arg Asn Gln His Asn Lys His His Ala Thr Pro 195 200 205 Gln Lys Leu Gln His Asp Val Asp Leu Asp Thr Leu Pro Leu Val Ala 210 215 220 Phe His Glu Arg Ile Ala Ala Lys Val Lys Ser Pro Ala Met Lys Ala 225 230 235 240 Trp Leu Ser Met Gln Ala Lys Leu Phe Ala Pro Val Thr Thr Leu Leu 245 250 255 Val Ala Leu Gly Trp Gln Leu Tyr Leu His Pro Arg His Met Leu Arg 260 265 270 Thr Lys His Tyr Asp Glu Leu Ala Met Leu Gly Ile Arg Tyr Gly Leu 275 280 285 Val Gly Tyr Leu Ala Ala Asn Tyr Gly Ala Gly Tyr Val Leu Ala Cys 290 295 300 Tyr Leu Leu Tyr Val Gln Leu Gly Ala Met Tyr Ile Phe Cys Asn Phe 305 310 315 320 Ala Val Ser His Thr His Leu Pro Val Val Glu Pro Asn Glu His Ala 325 330 335 Thr Trp Val Glu Tyr Ala Ala Asn His Thr Thr Asn Cys Ser Pro Ser 340 345 350 Trp Trp Cys Asp Trp Trp Met Ser Tyr Leu Asn Tyr Gln Ile Glu His 355 360 365 His Leu Tyr Pro Ser Met Pro Gln Phe Arg His Pro Lys Ile Ala Pro 370 375 380 Arg Val Lys Gln Leu Phe Glu Lys His Gly Leu His Tyr Asp Val Arg 385 390 395 400 Gly Tyr Phe Glu Ala Met Ala Asp Thr Phe Ala Asn Leu Asp Asn Val 405 410 415 Ala His Ala Pro Glu Lys Lys Met Gln 420 425 311329DNAPyramimonas cordata 31atgggaaagg gaggcaatgc tagcgctcct actgcgaaga aggaggtgtt gatcgagggg 60aagttttacg atgtcaccga cttcaggcac cccggtggtt cgatcatcaa gtttctctcg 120ggttctggtg ctgacgccac cgcttcctac cgcgagttcc acgttaggtc agcgaaggca 180gacaagttct tgaagacgct gccctcccgc gaagccactc cccaggagct gaagcaggcg 240gttgagttct ccaagctcaa cccgccctcc gcggagagtg cctctgctcc cctgaccgac 300cttgccaagg tggaagcgct gaacaaggac ttcgaggctt tccgtgagca gctcattcag 360gagggcttct ttaagcccaa tatcccgcat gtggtcaagc gcatcacgga agtcgtggcg 420atgatggccg tagcctcctg gatgatggtg cagaccaacg ctcttgttgt gaccctcgga 480gttctgatcc gcggcattgc acagggccgg tgcggttggc ttatgcacga gggcggccac 540tatagtctta ctgggaagat ctccattgat aggcgtctgc aggagtcaat ttacggattc 600ggctgtggaa tgtccggcgc ctggtggcgc aaccagcaca acaagcacca cgcaacccca 660cagaagctgc agcatgacgt cgacctggag acccttcctc tgatggcttt caacaacgct 720gttaccgata gacgcaaggt gaagcctggt agtctccagg ctctgtggct caagtaccag 780gccttcctct tcttccccgt gacctccctt ctggtcggcc tcggttggac caccgtcctc 840caccccaggc acagcttgcg caccaagcac tatttcgagc tgctctgcat ggctgctcgt 900tacgcgagtt tcgctgctct tttcgctccc aagtacggac ttgcaggagc tgccgggctc 960tacctcgcca ccttcgctgt cgggtgcaac tatattttca tcaacttctc ggtctctcac 1020actcacctgc ccgtgagcgg tgcgagcgag tacctgcatt gggtcgtgta ttcggccatc 1080cacaccacta acatcaaatc cagcatgctg tgcgattggt ggatgtcatt cctcaacttc 1140cagatcgagc atcacctgtt cccttcaatg ccccagttcc gccacaagat tatctccccg 1200cgtgtaaagg ccttgtttga gaagcacggt cttgtgtatg atgtgcgccc ctattggggg 1260gccatggctg acaccttcaa gaacttgaat gacgttggca ctcacgcatc tcactccaag 1320gcgcactag 132932442PRTPyramimonas cordata 32Met Gly Lys Gly Gly Asn Ala Ser Ala Pro Thr Ala Lys Lys Glu Val 1 5 10 15 Leu Ile Glu Gly Lys Phe Tyr Asp Val Thr Asp Phe Arg His Pro Gly 20 25 30 Gly Ser Ile Ile Lys Phe Leu Ser Gly Ser Gly Ala Asp Ala Thr Ala 35 40 45 Ser Tyr Arg Glu Phe His Val Arg Ser Ala Lys Ala Asp Lys Phe Leu 50 55 60 Lys Thr Leu Pro Ser Arg Glu Ala Thr Pro Gln Glu Leu Lys Gln Ala 65 70 75 80 Val Glu Phe Ser Lys Leu Asn Pro Pro Ser Ala Glu Ser Ala Ser Ala 85 90 95 Pro Leu Thr Asp Leu Ala Lys Val Glu Ala Leu Asn Lys Asp Phe Glu 100 105 110 Ala Phe Arg Glu Gln Leu Ile Gln Glu Gly Phe Phe Lys Pro Asn Ile 115 120 125 Pro His Val Val Lys Arg Ile Thr Glu Val Val Ala Met Met Ala Val 130 135 140 Ala Ser Trp Met Met Val Gln Thr Asn Ala Leu Val Val Thr Leu Gly 145 150 155 160 Val Leu Ile Arg Gly Ile Ala Gln Gly Arg Cys Gly Trp Leu Met His 165 170 175 Glu Gly Gly His Tyr Ser Leu Thr Gly Lys Ile Ser Ile Asp Arg Arg 180 185 190 Leu Gln Glu Ser Ile Tyr Gly Phe Gly Cys Gly Met Ser Gly Ala Trp 195 200 205 Trp Arg Asn Gln His Asn Lys His His Ala Thr Pro Gln Lys Leu Gln 210 215 220 His Asp Val Asp Leu Glu Thr Leu Pro Leu Met Ala Phe Asn Asn Ala 225 230 235 240 Val Thr Asp Arg Arg Lys Val Lys Pro Gly Ser Leu Gln Ala Leu Trp 245 250 255 Leu Lys Tyr Gln Ala Phe Leu Phe Phe Pro Val Thr Ser Leu Leu Val 260 265 270 Gly Leu Gly Trp Thr Thr Val Leu His Pro Arg His Ser Leu Arg Thr 275 280 285 Lys His Tyr Phe Glu Leu Leu Cys Met Ala Ala Arg Tyr Ala Ser Phe 290 295 300 Ala Ala Leu Phe Ala Pro Lys Tyr Gly Leu Ala Gly Ala Ala Gly Leu 305 310 315 320 Tyr Leu Ala Thr Phe Ala Val Gly Cys Asn Tyr Ile Phe Ile Asn Phe 325 330 335 Ser Val Ser His Thr His Leu Pro Val Ser Gly Ala Ser Glu Tyr Leu 340 345 350 His Trp Val Val Tyr Ser Ala Ile His Thr Thr Asn Ile Lys Ser Ser 355 360 365 Met Leu Cys Asp Trp Trp Met Ser Phe Leu Asn Phe Gln Ile Glu His 370 375 380 His Leu Phe Pro Ser Met Pro Gln Phe Arg His Lys Ile Ile Ser Pro 385 390 395 400 Arg Val Lys Ala Leu Phe Glu Lys His Gly Leu Val Tyr Asp Val Arg 405 410 415 Pro Tyr Trp Gly Ala Met Ala Asp Thr Phe Lys Asn Leu Asn Asp Val 420 425 430 Gly Thr His Ala Ser His Ser Lys Ala His 435 440 33804DNAPyramimonas cordata 33atggcgtcta ttgcgattcc ggctgcgctg gcagggactc ttggttatgt gacgtacaat 60gtcgcaaacc cagatattcc tgcatccgag aaggtgcctg cttactttat gcaggtcgag 120tattgggggc caacgattgg gaccatcggt tatcttctgt tcatctactt tggtaaacgg 180attatgcaaa acaggagcca gccgtttggc ctgaagaacg ctatgctggt gtacaacttc 240tatcagactt tcttcaactc gtactgcata tacctttttg tcacgtcgca ccgcgctcag 300gggctgaaag tttggggaaa catccccgat atgactgcca acagctgggg gatctcacag 360gtgatctggc tgcactacaa caacaagtac gttgagctgc tggacacgtt cttcatggtc 420atgcgcaaga agtttgacca gctttcgttc ctgcacattt accatcatac cctgttgatc 480tggtcttggt tcgtggtgat gaaattggag cccgttgggg actgctactt tggctctagc 540gtcaacacgt ttgtgcacgt cattatgtac tcgtactatg gccttgccgc gctcggggtg 600aattgcttct ggaagaagta cattacgcag attcagatgc tgcagttctg tatctgcgct 660tcgcactcga tttataccgc ctatgtgcag aacaccgcgt tctggttgcc ttacttgcag 720ctgtgggtga tggtgaacat gttcgtgttg ttcgccaact tctatcgcaa gcgctacaag 780agcaagggtg ccaagaagca

gtaa 80434807DNAArtificial SequenceCodon-optimized open reading frame for expression of Pyramimonas cordata 5-elongase in plants (version 1) 34atggcctcta tcgctatccc tgctgctctt gctggaactc ttggatacgt tacctacaat 60gtggctaacc ctgatatccc agcttctgag aaagttcctg cttacttcat gcaggttgag 120tactggggac ctactatcgg aactattgga tacctcctct tcatctactt cggaaagcgt 180atcatgcaga acagatctca acctttcgga ctcaagaacg ctatgctcgt ttacaacttc 240taccagacct tcttcaacag ctactgcatc taccttttcg ttacttctca tagggctcag 300ggacttaagg tttggggaaa catccctgat atgactgcta actcttgggg aatctctcag 360gttatctggc ttcactacaa caacaagtac gttgagcttc tcgacacctt cttcatggtg 420atgaggaaga agttcgacca gctttctttc cttcacatct accaccacac tcttctcatc 480tggtcatggt tcgttgttat gaagcttgag cctgttggag attgctactt cggatcttct 540gttaacacct tcgtgcacgt gatcatgtac tcttactacg gacttgctgc tcttggagtt 600aactgtttct ggaagaagta catcacccag atccagatgc ttcagttctg tatctgtgct 660tctcactcta tctacaccgc ttacgttcag aataccgctt tctggcttcc ttaccttcaa 720ctctgggtta tggtgaacat gttcgttctc ttcgccaact tctaccgtaa gaggtacaag 780tctaagggtg ctaagaagca gtgataa 80735867DNAArtificial SequenceCodon-optimized open reading frame for expression of Pyramimonas cordata 5-elongase in plants (version 2) 35atggaatttg ctcaacctct cgttgctatg gctcaagagc agtacgctgc tatcgatgct 60gttgttgctc ctgctatctt ctctgctacc gactctattg gatggggact caagcctatc 120tcttctgcta ctaaggatct ccctctcgtt gaatctccta cccctcttat cctttctctc 180ctcgcttact tcgctatcgt tggttctgga ctcgtttacc gtaaagtgtt ccctagaacc 240gttaagggac aggatccttt ccttctcaag gctcttatgc tcgctcacaa cgttttcctt 300atcggactca gcctttacat gtgcctcaag ctcgtttacg aggcttacgt gaacaagtac 360tccttctggg gaaacgctta caaccctgct caaaccgaga tggctaaggt gatctggatc 420ttctacgtgt ccaagatcta cgagttcatg gacaccttca tcatgcttct caagggaaac 480gttaaccagg tttccttcct ccatgtttac caccacggat ctatctctgg aatctggtgg 540atgatcactt atgctgctcc aggtggagat gcttacttct ctgctgctct caactcttgg 600gttcatgtgt gcatgtacac ctactacttc atggctgctg ttcttcctaa ggacgaaaag 660accaagagaa agtacctttg gtggggaaga taccttaccc agatgcaaat gttccagttc 720ttcatgaacc ttctccaggc tgtttacctc ctctactctt cttctcctta ccctaagttc 780attgctcaac tcctcgttgt ttacatggtt accctcctca tgcttttcgg aaacttctac 840tacatgaagc accacgcttc taagtga 86736807DNAArtificial SequenceCodon-optimized open reading frame for expression of Pyramimonas cordata 5-elongase in plants (version 3) 36atggcttcta tcgctatccc tgctgctctt gctggaactc ttggatacgt gacctacaac 60gtggctaacc ctgatattcc tgcttctgag aaggttccag cttacttcat gcaagtggag 120tactggggac ctactatcgg aactatcggt tacctcctct tcatctactt cggaaagcgt 180atcatgcaaa acagaagcca gcctttcgga cttaagaacg ctatgctcgt gtacaacttc 240taccagacct tcttcaacag ctactgcatc tacctcttcg ttacctctca tagggctcag 300ggacttaaag tttggggaaa catccctgat atgaccgcta actcttgggg aatctctcag 360gttatctggc tccactacaa caacaagtac gtggagcttc tcgatacctt cttcatggtg 420atgaggaaga agttcgacca gctttctttc cttcacatct accaccacac tcttctcatc 480tggtcatggt tcgtggttat gaagctcgag cctgttggag attgctactt cggatctagc 540gttaacacct tcgtgcacgt gatcatgtac tcttactacg gacttgctgc tcttggagtt 600aactgcttct ggaagaagta catcacccag atccagatgc ttcagttctg tatctgcgct 660tctcactcta tctacaccgc ttacgttcag aacactgctt tctggcttcc ttaccttcag 720ctctgggtga tggttaacat gttcgtgctc ttcgctaact tctaccgtaa aaggtacaag 780agcaagggtg ctaagaagca gtgataa 80737267PRTPyramimonas cordata 37Met Ala Ser Ile Ala Ile Pro Ala Ala Leu Ala Gly Thr Leu Gly Tyr 1 5 10 15 Val Thr Tyr Asn Val Ala Asn Pro Asp Ile Pro Ala Ser Glu Lys Val 20 25 30 Pro Ala Tyr Phe Met Gln Val Glu Tyr Trp Gly Pro Thr Ile Gly Thr 35 40 45 Ile Gly Tyr Leu Leu Phe Ile Tyr Phe Gly Lys Arg Ile Met Gln Asn 50 55 60 Arg Ser Gln Pro Phe Gly Leu Lys Asn Ala Met Leu Val Tyr Asn Phe 65 70 75 80 Tyr Gln Thr Phe Phe Asn Ser Tyr Cys Ile Tyr Leu Phe Val Thr Ser 85 90 95 His Arg Ala Gln Gly Leu Lys Val Trp Gly Asn Ile Pro Asp Met Thr 100 105 110 Ala Asn Ser Trp Gly Ile Ser Gln Val Ile Trp Leu His Tyr Asn Asn 115 120 125 Lys Tyr Val Glu Leu Leu Asp Thr Phe Phe Met Val Met Arg Lys Lys 130 135 140 Phe Asp Gln Leu Ser Phe Leu His Ile Tyr His His Thr Leu Leu Ile 145 150 155 160 Trp Ser Trp Phe Val Val Met Lys Leu Glu Pro Val Gly Asp Cys Tyr 165 170 175 Phe Gly Ser Ser Val Asn Thr Phe Val His Val Ile Met Tyr Ser Tyr 180 185 190 Tyr Gly Leu Ala Ala Leu Gly Val Asn Cys Phe Trp Lys Lys Tyr Ile 195 200 205 Thr Gln Ile Gln Met Leu Gln Phe Cys Ile Cys Ala Ser His Ser Ile 210 215 220 Tyr Thr Ala Tyr Val Gln Asn Thr Ala Phe Trp Leu Pro Tyr Leu Gln 225 230 235 240 Leu Trp Val Met Val Asn Met Phe Val Leu Phe Ala Asn Phe Tyr Arg 245 250 255 Lys Arg Tyr Lys Ser Lys Gly Ala Lys Lys Gln 260 265 381344DNAPavlova salina 38atgcctccga gcgcggcgaa gcagatgggc gcgagcacgg gcgtgcatgc gggcgtcaca 60gattcgtcgg ccttcacgcg caaggatgtc gccgacaggc cggacctcac gatcgtgggt 120gacagcgtgt acgatgcgaa ggcgttccgc tccgagcatc cgggtggcgc gcactttgtg 180tcgctgttcg gcgggcgcga tgccacggag gcgttcatgg agtaccaccg gcgcgcctgg 240cccaagtcgc gcatgtcgcg cttccacgtc ggctctctgg catcgaccga ggagcccgtc 300gccgccgatg agggctacct ccagctgtgc gctcgcatcg ccaagatggt gccgtcggtc 360agcagcgggt tcgcgccggc gtcgtactgg gtgaaggccg ggctgatcct cggctccgcg 420atcgcgctcg aggcgtacat gctgtacgcg ggcaagcgcc tgctcccgtc gatcgtgctc 480gggtggctgt ttgcgctgat tggcctgaac atccagcacg atgccaacca cggcgcgctc 540tccaagtcgg cctcggtcaa cctggcgctc gggttgtgcc aggactggat cggcgggagc 600atgatcctct ggctgcagga gcacgttgtc atgcaccact tgcacaccaa cgacgttgac 660aaggacccgg accagaaggc gcacggcgcc ctgcggctca agccgaccga cgcgtggagc 720ccgatgcact ggctgcagca cctctacctg ctgcctgggg agacgatgta cgccttcaag 780ctgctgtttc tcgacatcag cgagctggtg atgtggcggt gggagggcga gcccatcagc 840aagctggccg ggtacctctt catgccctcg ctgctcctca agctcacctt ctgggcgcgc 900tttgtcgcgc tgccgctgta cctcgcgccc agcgtgcaca cggcggtgtg catcgcggcg 960acggtaatga cggggagctt ctacctcgcc ttcttcttct tcatctcgca caacttcgag 1020ggcgtggcga gcgtcggacc ggacggcagc atcaccagca tgacgcgcgg cgcatccttc 1080ctcaagcggc aggccgagac ctcgtccaac gtgggcggcc cgctgctcgc cacgctcaac 1140ggcggcctca actaccaaat cgagcaccac ctcttcccca gggtgcacca cggcttctac 1200cctcgcctcg cgccgttggt caaggcggag ctcgaggcgc gcggcattga gtacaagcac 1260taccccacca tatggagcaa cctggcatcc acgctgaggc acatgtacgc gctcggccgc 1320aggccgcgca gcaaggcgga gtga 1344391347DNAArtificial SequenceCodon-optimized open reading frame for expression of Pavlova salina 4-desaturase in plants (version 1) 39atgccaccta gcgctgctaa gcaaatggga gcttctactg gtgttcatgc tggtgttact 60gactcttctg ctttcaccag aaaggatgtt gctgatagac ctgatctcac catcgttgga 120gattctgttt acgatgctaa ggctttcaga tctgagcatc ctggtggtgc tcatttcgtt 180tctttgttcg gaggaagaga tgctactgag gctttcatgg aataccatag aagggcttgg 240cctaagtcta gaatgtctag attccacgtt ggatctcttg cttctactga ggaacctgtt 300gctgctgatg agggatacct tcaactttgt gctaggatcg ctaagatggt gccttctgtt 360tcttctggat tcgctcctgc ttcttactgg gttaaggctg gacttatcct tggatctgct 420atcgctcttg aggcttacat gctttacgct ggaaagagac ttctcccttc tatcgttctt 480ggatggcttt tcgctcttat cggtcttaac atccagcatg atgctaacca tggtgctttg 540tctaagtctg cttctgttaa ccttgctctt ggactttgtc aggattggat cggaggatct 600atgatccttt ggcttcaaga gcatgttgtt atgcaccacc tccacactaa cgatgttgat 660aaggatcctg atcaaaaggc tcacggtgct cttagactca agcctactga tgcttggtca 720cctatgcatt ggcttcagca tctttacctt ttgcctggtg agactatgta cgctttcaag 780cttttgttcc tcgacatctc tgagcttgtt atgtggcgtt gggagggtga gcctatctct 840aagcttgctg gatacctctt tatgccttct ttgcttctca agcttacctt ctgggctaga 900ttcgttgctt tgcctcttta ccttgctcct tctgttcata ctgctgtgtg tatcgctgct 960actgttatga ctggatcttt ctacctcgct ttcttcttct tcatctccca caacttcgag 1020ggtgttgctt ctgttggacc tgatggatct atcacttcta tgactagagg tgctagcttc 1080cttaagagac aagctgagac ttcttctaac gttggaggac ctcttcttgc tactcttaac 1140ggtggactca actaccaaat tgagcatcac ttgttcccta gagttcacca tggattctac 1200cctagacttg ctcctcttgt taaggctgag cttgaggcta gaggaatcga gtacaagcac 1260taccctacta tctggtctaa ccttgcttct accctcagac atatgtacgc tcttggaaga 1320aggcctagat ctaaggctga gtaatga 1347401347DNAArtificial SequenceCodon-optimized open reading frame for expression of Pavlova salina 4-desaturase in plants (version 2) 40atgcctccat ctgctgctaa acagatggga gcttctactg gtgttcacgc tggtgttacc 60gattcttctg ctttcaccag aaaggatgtg gctgatagac ctgatcttac catcgttggt 120gactctgtgt acgatgctaa ggctttcaga tctgagcatc ctggtggtgc tcatttcgtt 180tcactcttcg gaggaagaga tgctactgag gctttcatgg aataccacag aagagcttgg 240cctaagtcta ggatgtctag gttccatgtt ggatctcttg cttctaccga ggaacctgtt 300gctgctgatg agggatacct tcagctttgt gctaggatcg ctaagatggt gccttctgtg 360tcatctggat tcgctccagc ttcttactgg gttaaggctg gacttatcct cggatctgct 420atcgctcttg aggcttacat gctctacgct ggaaagagac ttctcccttc tatcgttctt 480ggatggctct tcgctcttat cggacttaac atccagcatg acgctaacca tggtgctttg 540tctaagtctg ctagcgttaa ccttgctctt ggactttgtc aggattggat cggaggatct 600atgatccttt ggctccaaga gcatgttgtt atgcaccacc tccacaccaa cgatgttgat 660aaggaccctg atcaaaaggc tcatggtgct cttagactca agcctaccga tgcttggtca 720cctatgcatt ggcttcagca cctttacctt ctccctggtg aaactatgta cgctttcaag 780ctcctcttcc tcgatatctc tgagcttgtg atgtggagat gggagggtga acctatctct 840aagctcgctg gatacctctt catgccttct cttctcctca agcttacctt ctgggctaga 900ttcgttgctc ttcctcttta cctcgctcct tctgttcata ctgctgtgtg tatcgctgct 960actgttatga ccggaagctt ctaccttgct ttcttcttct tcatcagcca caacttcgag 1020ggtgttgctt ctgttggacc tgatggatct atcacctcta tgaccagggg agcttctttc 1080cttaagaggc aggctgagac ttcttctaat gtgggaggac ctcttcttgc tactcttaac 1140ggtggactca actaccaaat cgagcaccac cttttcccta gagttcacca cggattctac 1200cctagacttg ctcctcttgt gaaggctgaa cttgaggcta gaggaatcga gtacaagcac 1260taccctacca tctggtctaa cctcgcttct accctcagac atatgtacgc tcttggaaga 1320aggcctagat ctaaggctga gtgatga 134741447PRTPavlova salina 41Met Pro Pro Ser Ala Ala Lys Gln Met Gly Ala Ser Thr Gly Val His 1 5 10 15 Ala Gly Val Thr Asp Ser Ser Ala Phe Thr Arg Lys Asp Val Ala Asp 20 25 30 Arg Pro Asp Leu Thr Ile Val Gly Asp Ser Val Tyr Asp Ala Lys Ala 35 40 45 Phe Arg Ser Glu His Pro Gly Gly Ala His Phe Val Ser Leu Phe Gly 50 55 60 Gly Arg Asp Ala Thr Glu Ala Phe Met Glu Tyr His Arg Arg Ala Trp 65 70 75 80 Pro Lys Ser Arg Met Ser Arg Phe His Val Gly Ser Leu Ala Ser Thr 85 90 95 Glu Glu Pro Val Ala Ala Asp Glu Gly Tyr Leu Gln Leu Cys Ala Arg 100 105 110 Ile Ala Lys Met Val Pro Ser Val Ser Ser Gly Phe Ala Pro Ala Ser 115 120 125 Tyr Trp Val Lys Ala Gly Leu Ile Leu Gly Ser Ala Ile Ala Leu Glu 130 135 140 Ala Tyr Met Leu Tyr Ala Gly Lys Arg Leu Leu Pro Ser Ile Val Leu 145 150 155 160 Gly Trp Leu Phe Ala Leu Ile Gly Leu Asn Ile Gln His Asp Ala Asn 165 170 175 His Gly Ala Leu Ser Lys Ser Ala Ser Val Asn Leu Ala Leu Gly Leu 180 185 190 Cys Gln Asp Trp Ile Gly Gly Ser Met Ile Leu Trp Leu Gln Glu His 195 200 205 Val Val Met His His Leu His Thr Asn Asp Val Asp Lys Asp Pro Asp 210 215 220 Gln Lys Ala His Gly Ala Leu Arg Leu Lys Pro Thr Asp Ala Trp Ser 225 230 235 240 Pro Met His Trp Leu Gln His Leu Tyr Leu Leu Pro Gly Glu Thr Met 245 250 255 Tyr Ala Phe Lys Leu Leu Phe Leu Asp Ile Ser Glu Leu Val Met Trp 260 265 270 Arg Trp Glu Gly Glu Pro Ile Ser Lys Leu Ala Gly Tyr Leu Phe Met 275 280 285 Pro Ser Leu Leu Leu Lys Leu Thr Phe Trp Ala Arg Phe Val Ala Leu 290 295 300 Pro Leu Tyr Leu Ala Pro Ser Val His Thr Ala Val Cys Ile Ala Ala 305 310 315 320 Thr Val Met Thr Gly Ser Phe Tyr Leu Ala Phe Phe Phe Phe Ile Ser 325 330 335 His Asn Phe Glu Gly Val Ala Ser Val Gly Pro Asp Gly Ser Ile Thr 340 345 350 Ser Met Thr Arg Gly Ala Ser Phe Leu Lys Arg Gln Ala Glu Thr Ser 355 360 365 Ser Asn Val Gly Gly Pro Leu Leu Ala Thr Leu Asn Gly Gly Leu Asn 370 375 380 Tyr Gln Ile Glu His His Leu Phe Pro Arg Val His His Gly Phe Tyr 385 390 395 400 Pro Arg Leu Ala Pro Leu Val Lys Ala Glu Leu Glu Ala Arg Gly Ile 405 410 415 Glu Tyr Lys His Tyr Pro Thr Ile Trp Ser Asn Leu Ala Ser Thr Leu 420 425 430 Arg His Met Tyr Ala Leu Gly Arg Arg Pro Arg Ser Lys Ala Glu 435 440 445 42792DNAIsochrysis galbana 42atggccctcg caaacgacgc gggagagcgc atctgggcgg ctgtgaccga cccggaaatc 60ctcattggca ccttctcgta cttgctactc aaaccgctgc tccgcaattc cgggctggtg 120gatgagaaga agggcgcata caggacgtcc atgatctggt acaacgttct gctggcgctc 180ttctctgcgc tgagcttcta cgtgacggcg accgccctcg gctgggacta tggtacgggc 240gcgtggctgc gcaggcaaac cggcgacaca ccgcagccgc tcttccagtg cccgtccccg 300gtttgggact cgaagctctt cacatggacc gccaaggcat tctattactc caagtacgtg 360gagtacctcg acacggcctg gctggtgctc aagggcaaga gggtctcctt tctccaggcc 420ttccaccact ttggcgcgcc gtgggatgtg tacctcggca ttcggctgca caacgagggc 480gtatggatct tcatgttttt caactcgttc attcacacca tcatgtacac ctactacggc 540ctcaccgccg ccgggtataa gttcaaggcc aagccgctca tcaccgcgat gcagatctgc 600cagttcgtgg gcggcttcct gttggtctgg gactacatca acgtcccctg cttcaactcg 660gacaaaggga agttgttcag ctgggctttc aactatgcat acgtcggctc ggtcttcttg 720ctcttctgcc actttttcta ccaggacaac ttggcaacga agaaatcggc caaggcgggc 780aagcagctct ag 79243263PRTIsochrysis galbana 43Met Ala Leu Ala Asn Asp Ala Gly Glu Arg Ile Trp Ala Ala Val Thr 1 5 10 15 Asp Pro Glu Ile Leu Ile Gly Thr Phe Ser Tyr Leu Leu Leu Lys Pro 20 25 30 Leu Leu Arg Asn Ser Gly Leu Val Asp Glu Lys Lys Gly Ala Tyr Arg 35 40 45 Thr Ser Met Ile Trp Tyr Asn Val Leu Leu Ala Leu Phe Ser Ala Leu 50 55 60 Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly Trp Asp Tyr Gly Thr Gly 65 70 75 80 Ala Trp Leu Arg Arg Gln Thr Gly Asp Thr Pro Gln Pro Leu Phe Gln 85 90 95 Cys Pro Ser Pro Val Trp Asp Ser Lys Leu Phe Thr Trp Thr Ala Lys 100 105 110 Ala Phe Tyr Tyr Ser Lys Tyr Val Glu Tyr Leu Asp Thr Ala Trp Leu 115 120 125 Val Leu Lys Gly Lys Arg Val Ser Phe Leu Gln Ala Phe His His Phe 130 135 140 Gly Ala Pro Trp Asp Val Tyr Leu Gly Ile Arg Leu His Asn Glu Gly 145 150 155 160 Val Trp Ile Phe Met Phe Phe Asn Ser Phe Ile His Thr Ile Met Tyr 165 170 175 Thr Tyr Tyr Gly Leu Thr Ala Ala Gly Tyr Lys Phe Lys Ala Lys Pro 180 185 190 Leu Ile Thr Ala Met Gln Ile Cys Gln Phe Val Gly Gly Phe Leu Leu 195 200 205 Val Trp Asp Tyr Ile Asn Val Pro Cys Phe Asn Ser Asp Lys Gly Lys 210 215 220 Leu Phe Ser Trp Ala Phe Asn Tyr Ala Tyr Val Gly Ser Val Phe Leu 225 230 235 240 Leu Phe Cys His Phe Phe Tyr Gln Asp Asn Leu Ala Thr Lys Lys Ser 245 250 255 Ala Lys Ala Gly Lys Gln Leu 260 44801DNAEmiliania huxleyi 44atgctcgatc gcgcctcgtc cgacgcggcc atctggtctg cggtgtccga tccggaaatc 60ctgatcggca ctttctccta cctgctgctc aagccgctgc tacgcaactc agggctcgtg 120gacgagcgga aaggcgccta ccggacctcg atgatctggt acaacgtggt gctcgcgctc 180ttctccgcga cgagcttcta cgtgactgcg accgcgctcg ggtgggacaa gggcaccggc 240gagtggctcc gcagtctcac gggcgacagc ccgcagcagc tgtggcaatg cccgtcgagg 300gtatgggact ccaagctgtt

cctgtggacg gccaaggcct tctactactc aaagtacgtg 360gagtacctcg acacggcgtg gctcgtcctc aaggggaaga aggtctcctt cctgcagggc 420ttccaccact ttggcgcgcc gtgggacgtg tacctgggca ttcggctgaa gaacgagggc 480gtgtggatct tcatgttctt caactcgttc atccacacgg tcatgtacac gtactacggc 540ctcaccgccg cgggctacaa gatccgcggc aagccgatca tcaccgcgat gcaaataagc 600cagttcgtcg gcggctttgt cctagtgtgg gactacatca acgtgccgtg cttccacgcc 660gacgccgggc aggtcttcag ctgggtcttt aactatgctt acgtcggctc cgtctttctg 720ctgttctgcc acttcttcta catggacaac atcgcgaagg ccaaggccaa gaaggccgtc 780gctacccgca aggcgctgtg a 80145801DNAArtificial SequenceCodon-optimized open reading frame for expression of Emiliania huxleyi 9-elongase in plants 45atgcttgata gagcttcatc tgatgctgct atttggagcg ctgtttctga tcctgagatc 60cttatcggaa ccttctctta ccttttgctt aagcctctcc tcagaaactc tggacttgtg 120gatgagagaa agggagctta ccgtacttct atgatctggt acaacgttgt tcttgctctt 180ttctctgcta cctctttcta cgttactgct actgctcttg gatgggataa gggaactggt 240gagtggctta gatctcttac tggtgattct cctcaacaac tttggcagtg cccttctaga 300gtttgggaca gcaaactctt cttgtggact gctaaagcct tctactactc caagtacgtt 360gagtaccttg atactgcttg gcttgttctc aagggaaaga aggtttcatt cctccaggga 420ttccatcatt tcggtgctcc atgggatgtt taccttggaa tcaggcttaa gaacgaggga 480gtttggatct tcatgttctt caacagcttc atccacactg ttatgtacac ttactacgga 540cttactgctg ctggatacaa gatcagagga aagcctatca tcaccgctat gcaaatctct 600caattcgttg gtggattcgt tcttgtgtgg gactacatca acgttccttg tttccatgct 660gatgctggac aagttttctc ttgggtgttc aactacgctt atgtgggatc tgttttcctt 720cttttctgcc acttcttcta catggacaac attgctaagg ctaaggctaa aaaggctgtt 780gctaccagaa aggctctttg a 80146266PRTEmiliania huxleyi 46Met Leu Asp Arg Ala Ser Ser Asp Ala Ala Ile Trp Ser Ala Val Ser 1 5 10 15 Asp Pro Glu Ile Leu Ile Gly Thr Phe Ser Tyr Leu Leu Leu Lys Pro 20 25 30 Leu Leu Arg Asn Ser Gly Leu Val Asp Glu Arg Lys Gly Ala Tyr Arg 35 40 45 Thr Ser Met Ile Trp Tyr Asn Val Val Leu Ala Leu Phe Ser Ala Thr 50 55 60 Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly Trp Asp Lys Gly Thr Gly 65 70 75 80 Glu Trp Leu Arg Ser Leu Thr Gly Asp Ser Pro Gln Gln Leu Trp Gln 85 90 95 Cys Pro Ser Arg Val Trp Asp Ser Lys Leu Phe Leu Trp Thr Ala Lys 100 105 110 Ala Phe Tyr Tyr Ser Lys Tyr Val Glu Tyr Leu Asp Thr Ala Trp Leu 115 120 125 Val Leu Lys Gly Lys Lys Val Ser Phe Leu Gln Gly Phe His His Phe 130 135 140 Gly Ala Pro Trp Asp Val Tyr Leu Gly Ile Arg Leu Lys Asn Glu Gly 145 150 155 160 Val Trp Ile Phe Met Phe Phe Asn Ser Phe Ile His Thr Val Met Tyr 165 170 175 Thr Tyr Tyr Gly Leu Thr Ala Ala Gly Tyr Lys Ile Arg Gly Lys Pro 180 185 190 Ile Ile Thr Ala Met Gln Ile Ser Gln Phe Val Gly Gly Phe Val Leu 195 200 205 Val Trp Asp Tyr Ile Asn Val Pro Cys Phe His Ala Asp Ala Gly Gln 210 215 220 Val Phe Ser Trp Val Phe Asn Tyr Ala Tyr Val Gly Ser Val Phe Leu 225 230 235 240 Leu Phe Cys His Phe Phe Tyr Met Asp Asn Ile Ala Lys Ala Lys Ala 245 250 255 Lys Lys Ala Val Ala Thr Arg Lys Ala Leu 260 265 47819DNAPavlova pinguis 47atggttgcgc cacccatcac gctcgagtgg ctgctttcgc cgaagctcaa ggatgcagtg 60ttcggtgggg aggtgctcta cttctccatt gcctacctgt ttcttgcgcc cattttgaag 120cgcaccccgt tggtggacac gcggaagggc gcgtataaga gtggtatgat cgcgtacaac 180gtgatcatgt gcgtgttctc gctggtgtgc ttcatctgcc agctcgcagc cctgggctat 240gacatgggct acttgcagtg ggtgcgtgac ctcacagggg acgagattgt ccccctctac 300caggacgtgt ccccgtcccc cgccttctcc aacaagctct tcaagtattc gtctattgcc 360ttccactact ccaagtatgt tgagtacatg gacaccgcat ggctggtgat gaagggcaag 420cccgtgtcct tgctccaggg cttccaccac tttggcgccg cctgggacac ctactttggc 480atcaccttcc agaacgaggg catctacgtg ttcgtggtgc tcaacgcctt catccacacg 540atcatgtacg catactacgc ggccactgcg gcgggtctca agttctcact gaagttcgtc 600atcacgctca tgcagatcac ccaattcaac gtgggcttcg taatggtgta tcactacatc 660accctggagt acttccgcaa ctcaccggag ctcgtcttct cctacctttt caactatgcg 720tacgtctgca cggttctcct cctcttcatg cagttcttct acatggacaa ctttggcaag 780aagaaggccg ctgccgccgc gggcaagaag aagaagtag 81948272PRTPavlova pinguis 48Met Val Ala Pro Pro Ile Thr Leu Glu Trp Leu Leu Ser Pro Lys Leu 1 5 10 15 Lys Asp Ala Val Phe Gly Gly Glu Val Leu Tyr Phe Ser Ile Ala Tyr 20 25 30 Leu Phe Leu Ala Pro Ile Leu Lys Arg Thr Pro Leu Val Asp Thr Arg 35 40 45 Lys Gly Ala Tyr Lys Ser Gly Met Ile Ala Tyr Asn Val Ile Met Cys 50 55 60 Val Phe Ser Leu Val Cys Phe Ile Cys Gln Leu Ala Ala Leu Gly Tyr 65 70 75 80 Asp Met Gly Tyr Leu Gln Trp Val Arg Asp Leu Thr Gly Asp Glu Ile 85 90 95 Val Pro Leu Tyr Gln Asp Val Ser Pro Ser Pro Ala Phe Ser Asn Lys 100 105 110 Leu Phe Lys Tyr Ser Ser Ile Ala Phe His Tyr Ser Lys Tyr Val Glu 115 120 125 Tyr Met Asp Thr Ala Trp Leu Val Met Lys Gly Lys Pro Val Ser Leu 130 135 140 Leu Gln Gly Phe His His Phe Gly Ala Ala Trp Asp Thr Tyr Phe Gly 145 150 155 160 Ile Thr Phe Gln Asn Glu Gly Ile Tyr Val Phe Val Val Leu Asn Ala 165 170 175 Phe Ile His Thr Ile Met Tyr Ala Tyr Tyr Ala Ala Thr Ala Ala Gly 180 185 190 Leu Lys Phe Ser Leu Lys Phe Val Ile Thr Leu Met Gln Ile Thr Gln 195 200 205 Phe Asn Val Gly Phe Val Met Val Tyr His Tyr Ile Thr Leu Glu Tyr 210 215 220 Phe Arg Asn Ser Pro Glu Leu Val Phe Ser Tyr Leu Phe Asn Tyr Ala 225 230 235 240 Tyr Val Cys Thr Val Leu Leu Leu Phe Met Gln Phe Phe Tyr Met Asp 245 250 255 Asn Phe Gly Lys Lys Lys Ala Ala Ala Ala Ala Gly Lys Lys Lys Lys 260 265 270 49840DNAPavlova salina 49atggcgactg aagggatgcc ggcgataacg ctggactggc tgctctcgcc cgggctgaag 60gatgccgtaa ttggcgggga ggtgctctac ttttcgcttg ggtatctgct gctcgagccc 120atcctcaagc gctcaccgtt tgtggacaag cgcaagggcg cataccgcaa cggcatgatc 180gcgtacaaca tcctcatgtg cggtttctcg ctggtatgct tcgtgtgcca gatggcggcg 240ctcggccttg atcgcggcca cctgcagttt gtccgcgacc tcacgggcga cagcgtggtg 300cagctctacc aggacgtgag cccatcccct gcattcgcga acaagctctt ccggtactca 360gcggtggcgt tccactactc aaagtacgtg gagtacatgg acacagcgtg gcttgtgctg 420aagggcaagc ccgtctcgtt cctgcagggc ttccaccact tcggcgccgc gtgggacacc 480tactttggca tcacgtttca gaacgagggc acctacgtct ttgtgctgct caacgcattc 540atccacacaa tcatgtacac ctactacggc gcgacggcag cgggcatcaa aatctcgatg 600aagccgctga tcaccctcat gcagatcacg cagttcctgc tgggcttcgc gctcgtctac 660ccgtacattg acctcggcta cttccgtgcg tcgcccgagc tcgtgtggag ctacctgttc 720aactatgcgt acgtactcat ggtgctcttc ctcttcatgc gcttcttcta ccacgacaac 780tttagcaagc acaagccaat ctcgcgcatc gactccagca accgcatgaa aaccgagtag 84050279PRTPavlova salina 50Met Ala Thr Glu Gly Met Pro Ala Ile Thr Leu Asp Trp Leu Leu Ser 1 5 10 15 Pro Gly Leu Lys Asp Ala Val Ile Gly Gly Glu Val Leu Tyr Phe Ser 20 25 30 Leu Gly Tyr Leu Leu Leu Glu Pro Ile Leu Lys Arg Ser Pro Phe Val 35 40 45 Asp Lys Arg Lys Gly Ala Tyr Arg Asn Gly Met Ile Ala Tyr Asn Ile 50 55 60 Leu Met Cys Gly Phe Ser Leu Val Cys Phe Val Cys Gln Met Ala Ala 65 70 75 80 Leu Gly Leu Asp Arg Gly His Leu Gln Phe Val Arg Asp Leu Thr Gly 85 90 95 Asp Ser Val Val Gln Leu Tyr Gln Asp Val Ser Pro Ser Pro Ala Phe 100 105 110 Ala Asn Lys Leu Phe Arg Tyr Ser Ala Val Ala Phe His Tyr Ser Lys 115 120 125 Tyr Val Glu Tyr Met Asp Thr Ala Trp Leu Val Leu Lys Gly Lys Pro 130 135 140 Val Ser Phe Leu Gln Gly Phe His His Phe Gly Ala Ala Trp Asp Thr 145 150 155 160 Tyr Phe Gly Ile Thr Phe Gln Asn Glu Gly Thr Tyr Val Phe Val Leu 165 170 175 Leu Asn Ala Phe Ile His Thr Ile Met Tyr Thr Tyr Tyr Gly Ala Thr 180 185 190 Ala Ala Gly Ile Lys Ile Ser Met Lys Pro Leu Ile Thr Leu Met Gln 195 200 205 Ile Thr Gln Phe Leu Leu Gly Phe Ala Leu Val Tyr Pro Tyr Ile Asp 210 215 220 Leu Gly Tyr Phe Arg Ala Ser Pro Glu Leu Val Trp Ser Tyr Leu Phe 225 230 235 240 Asn Tyr Ala Tyr Val Leu Met Val Leu Phe Leu Phe Met Arg Phe Phe 245 250 255 Tyr His Asp Asn Phe Ser Lys His Lys Pro Ile Ser Arg Ile Asp Ser 260 265 270 Ser Asn Arg Met Lys Thr Glu 275 511284DNAPavlova salina 51atgggacgcg gcggagacag cagtgggcag gcgcatccgg cggcggagct ggcggtcccg 60agcgaccgcg cggaggtgag caacgctgac agcaaagcgc tgcacatcgt gctgtatggc 120aagcgcgtgg atgtgaccaa gttccaacgc acgcacccgg gtggtagcaa ggtcttccgg 180atcttccagg accgcgatgc gacggagcag ttcgagtcct accactcgaa gcgcgcgatc 240aagatgatgg agggcatgct caagaagtct gaggatgctc ccgccgacac gcccttgccc 300tcccagtcac cgatggggaa ggacttcaag gcgatgatcg agcggcacgt tgcagcgggt 360tactacgatc catgcccgct cgatgagctg ttcaagctca gcctcgtgct cctcccgacc 420tttgcgggca tgtacatgct caaggcgggc gtcggctccc cgctctgcgg cgccctcatg 480gtgagctttg gctggtacct cgatggctgg ctcgcgcacg actatctgca ccactccgtc 540ttcaaggggt ccgtcgcacg caccgtcggg tggaacaacg cggcgggcta cttcctcggc 600ttcgtgcagg ggtatgcggt cgagtggtgg cgcgcgcggc ataacacgca ccacgtgtgc 660accaatgagg acggctcgga ccccgacatc aaaacggcgc cgctgctcat atacgtgcgc 720aacaagccga gcatcgccaa gcgcctgaac gccttccagc gctaccagca gtactactat 780gtgccggtga tggcaatcct cgacctgtac tggcggctcg agtcgatcgc ctacgtcgcg 840atgcgcctgc cgaagatgct gccgcaggcc ctcgcactcg tcgcgcacta cgccatcgtc 900gcgtgggtct ttgcgggcaa ctaccacctg ctcccgctcg tgacggttct gcgcgggttt 960ggcactggga tcaccgtttt cgcgacgcac tacggtgagg acattctcga cgcggaccag 1020gtgcgtcaca tgacgctcgt cgagcagacg gcactcacct cgcgcaacat ctcgggcggc 1080tggctcgtga acgtgctcac cggcttcatc tcactgcaga cggagcacca cctgttcccg 1140atgatgccaa ccggcaacct catgactatc cagcccgagg tgcgcgcctt cttcaagaag 1200cacggacttg agtaccgcga gggcaacctc attgagtgcg tgcggcagaa catccgtgcg 1260cttgcattcg agcacctgct ttga 128452427PRTPavlova salina 52Met Gly Arg Gly Gly Asp Ser Ser Gly Gln Ala His Pro Ala Ala Glu 1 5 10 15 Leu Ala Val Pro Ser Asp Arg Ala Glu Val Ser Asn Ala Asp Ser Lys 20 25 30 Ala Leu His Ile Val Leu Tyr Gly Lys Arg Val Asp Val Thr Lys Phe 35 40 45 Gln Arg Thr His Pro Gly Gly Ser Lys Val Phe Arg Ile Phe Gln Asp 50 55 60 Arg Asp Ala Thr Glu Gln Phe Glu Ser Tyr His Ser Lys Arg Ala Ile 65 70 75 80 Lys Met Met Glu Gly Met Leu Lys Lys Ser Glu Asp Ala Pro Ala Asp 85 90 95 Thr Pro Leu Pro Ser Gln Ser Pro Met Gly Lys Asp Phe Lys Ala Met 100 105 110 Ile Glu Arg His Val Ala Ala Gly Tyr Tyr Asp Pro Cys Pro Leu Asp 115 120 125 Glu Leu Phe Lys Leu Ser Leu Val Leu Leu Pro Thr Phe Ala Gly Met 130 135 140 Tyr Met Leu Lys Ala Gly Val Gly Ser Pro Leu Cys Gly Ala Leu Met 145 150 155 160 Val Ser Phe Gly Trp Tyr Leu Asp Gly Trp Leu Ala His Asp Tyr Leu 165 170 175 His His Ser Val Phe Lys Gly Ser Val Ala Arg Thr Val Gly Trp Asn 180 185 190 Asn Ala Ala Gly Tyr Phe Leu Gly Phe Val Gln Gly Tyr Ala Val Glu 195 200 205 Trp Trp Arg Ala Arg His Asn Thr His His Val Cys Thr Asn Glu Asp 210 215 220 Gly Ser Asp Pro Asp Ile Lys Thr Ala Pro Leu Leu Ile Tyr Val Arg 225 230 235 240 Asn Lys Pro Ser Ile Ala Lys Arg Leu Asn Ala Phe Gln Arg Tyr Gln 245 250 255 Gln Tyr Tyr Tyr Val Pro Val Met Ala Ile Leu Asp Leu Tyr Trp Arg 260 265 270 Leu Glu Ser Ile Ala Tyr Val Ala Met Arg Leu Pro Lys Met Leu Pro 275 280 285 Gln Ala Leu Ala Leu Val Ala His Tyr Ala Ile Val Ala Trp Val Phe 290 295 300 Ala Gly Asn Tyr His Leu Leu Pro Leu Val Thr Val Leu Arg Gly Phe 305 310 315 320 Gly Thr Gly Ile Thr Val Phe Ala Thr His Tyr Gly Glu Asp Ile Leu 325 330 335 Asp Ala Asp Gln Val Arg His Met Thr Leu Val Glu Gln Thr Ala Leu 340 345 350 Thr Ser Arg Asn Ile Ser Gly Gly Trp Leu Val Asn Val Leu Thr Gly 355 360 365 Phe Ile Ser Leu Gln Thr Glu His His Leu Phe Pro Met Met Pro Thr 370 375 380 Gly Asn Leu Met Thr Ile Gln Pro Glu Val Arg Ala Phe Phe Lys Lys 385 390 395 400 His Gly Leu Glu Tyr Arg Glu Gly Asn Leu Ile Glu Cys Val Arg Gln 405 410 415 Asn Ile Arg Ala Leu Ala Phe Glu His Leu Leu 420 425 53172PRTTomato bushy stunt virus 53Met Glu Arg Ala Ile Gln Gly Asn Asp Ala Arg Glu Gln Ala Asn Ser 1 5 10 15 Glu Arg Trp Asp Gly Gly Ser Gly Gly Thr Thr Ser Pro Phe Lys Leu 20 25 30 Pro Asp Glu Ser Pro Ser Trp Thr Glu Trp Arg Leu His Asn Asp Glu 35 40 45 Thr Asn Ser Asn Gln Asp Asn Pro Leu Gly Phe Lys Glu Ser Trp Gly 50 55 60 Phe Gly Lys Val Val Phe Lys Arg Tyr Leu Arg Tyr Asp Arg Thr Glu 65 70 75 80 Ala Ser Leu His Arg Val Leu Gly Ser Trp Thr Gly Asp Ser Val Asn 85 90 95 Tyr Ala Ala Ser Arg Phe Phe Gly Phe Asp Gln Ile Gly Cys Thr Tyr 100 105 110 Ser Ile Arg Phe Arg Gly Val Ser Ile Thr Val Ser Gly Gly Ser Arg 115 120 125 Thr Leu Gln His Leu Cys Glu Met Ala Ile Arg Ser Lys Gln Glu Leu 130 135 140 Leu Gln Leu Ala Pro Ile Glu Val Glu Ser Asn Val Ser Arg Gly Cys 145 150 155 160 Pro Glu Gly Thr Glu Thr Phe Glu Lys Glu Ser Glu 165 170 54116PRTTomato yellow leaf curl virus 54Met Trp Asp Pro Leu Leu Asn Glu Phe Pro Glu Ser Val His Gly Phe 1 5 10 15 Arg Cys Met Leu Ala Ile Lys Tyr Leu Gln Ser Val Glu Glu Thr Tyr 20 25 30 Glu Pro Asn Thr Leu Gly His Asp Leu Ile Arg Asp Leu Ile Ser Val 35 40 45 Val Arg Ala Arg Asp Tyr Val Glu Ala Thr Arg Arg Tyr Asn His Phe 50 55 60 His Ala Arg Leu Glu Gly Ser Pro Lys Ala Glu Leu Arg Gln Pro Ile 65 70 75 80 Gln Gln Pro Cys Cys Cys Pro His Cys Pro Arg His Lys Gln Ala Thr 85 90 95 Ile Met Asp Val Gln Ala His Val Pro Glu Ala Gln Asn Ile Gln Asn 100 105 110 Val Ser Lys Pro 115 55351PRTTurnip crinkle virus 55Met Glu Asn Asp Pro Arg Val Arg Lys Phe Ala Ser Glu Gly Ala Gln 1 5 10 15 Trp Ala Ile Lys Trp Gln Lys Lys Gly Trp Ser Ser Leu Thr Ser Arg 20 25 30 Gln Lys Gln Thr Ala Arg Ala Ala Met Gly Ile Lys Leu Ser Pro Val 35 40 45 Ala Gln Pro Val Gln Lys

Val Thr Arg Leu Ser Ala Pro Val Ala Leu 50 55 60 Ala Tyr Arg Glu Val Ser Thr Gln Pro Arg Val Ser Thr Ala Arg Asp 65 70 75 80 Gly Ile Thr Arg Ser Gly Ser Glu Leu Ile Thr Thr Leu Lys Lys Asn 85 90 95 Thr Asp Thr Glu Pro Lys Tyr Thr Thr Ala Val Leu Asn Pro Ser Glu 100 105 110 Pro Gly Thr Phe Asn Gln Leu Ile Lys Glu Ala Ala Gln Tyr Glu Lys 115 120 125 Tyr Arg Phe Thr Ser Leu Arg Phe Arg Tyr Ser Pro Met Ser Pro Ser 130 135 140 Thr Thr Gly Gly Lys Val Ala Leu Ala Phe Asp Arg Asp Ala Ala Lys 145 150 155 160 Pro Pro Pro Asn Asp Leu Ala Ser Leu Tyr Asn Ile Glu Gly Cys Val 165 170 175 Ser Ser Val Pro Trp Thr Gly Phe Ile Leu Thr Val Pro Thr Asp Ser 180 185 190 Thr Asp Arg Phe Val Ala Asp Gly Ile Ser Asp Pro Lys Leu Val Asn 195 200 205 Phe Gly Lys Leu Ile Met Ala Thr Tyr Gly Gln Gly Ala Asn Asp Ala 210 215 220 Ala Gln Leu Gly Glu Val Arg Val Glu Tyr Thr Val Gln Leu Lys Asn 225 230 235 240 Arg Thr Gly Ser Thr Ser Asp Ala Gln Ile Gly Asp Phe Ala Gly Val 245 250 255 Lys Asp Gly Pro Arg Leu Val Ser Trp Ser Lys Thr Lys Gly Thr Ala 260 265 270 Gly Trp Glu His Asp Cys His Phe Leu Gly Thr Gly Asn Phe Ser Leu 275 280 285 Thr Leu Phe Tyr Glu Lys Ala Pro Val Ser Gly Leu Glu Asn Ala Asp 290 295 300 Ala Ser Asp Phe Ser Val Leu Gly Glu Ala Ala Ala Gly Ser Val Gln 305 310 315 320 Trp Ala Gly Val Lys Val Ala Glu Arg Gly Gln Ser Val Lys Met Val 325 330 335 Thr Thr Glu Glu Gln Pro Arg Gly Lys Trp Gln Ala Leu Arg Ile 340 345 350 56303PRTPea mosaic virus 56Met His Gly Ile Glu Gln Pro Gln Leu Pro Leu Asp Tyr Val His Arg 1 5 10 15 Cys Ala Ser Thr Ser Phe Leu Leu Ala Ser Leu Asp Gly Leu Leu Ser 20 25 30 Glu Ala Arg Glu Leu Ser Gly Pro Leu Ala Leu Ile Thr Ser Ser Tyr 35 40 45 Tyr Leu Leu Val Ser Ile Ala Leu Cys Trp Ala Ile Pro Gly Ser Phe 50 55 60 Trp Tyr Arg Pro Gly Cys Trp Leu Gln Pro Val Ser Gly Arg Asn Leu 65 70 75 80 Ile Phe Cys Gly Pro Thr Glu Ala Leu Gln Arg Phe Arg Leu Tyr Ala 85 90 95 Ala Arg Leu Gly Leu Val Leu Ser Glu Asn Cys Pro Arg His Gly Gln 100 105 110 Ser Ala Ala Ile Thr Leu Gln Ser Tyr Trp Ala Leu Pro Asn Asn Ile 115 120 125 Trp Met Asp Met Ala Gln Leu Asp Leu Leu Thr Phe Ser Met Pro Ile 130 135 140 Ala Asn Thr Phe Ala Tyr Leu Ala Asp Cys Glu Ala Arg Phe Pro Pro 145 150 155 160 Ile Val Glu Gly Val Gly Ser Ala Tyr Tyr Val Pro Thr Leu Leu Gly 165 170 175 Leu Thr His Gln Asp Pro Arg Leu Tyr Leu Ala Leu Arg Arg Arg Asn 180 185 190 Leu Asp Leu Ser Gly Glu Pro His Arg Val Arg Pro Gly Val Leu Glu 195 200 205 Ser Met Ala Leu Leu Cys Ser Ser Val Arg Ser Thr Ser Arg Ser Arg 210 215 220 Gln Ile Pro Pro Leu Tyr Gly Ser Val Leu His His Val Leu Gly Leu 225 230 235 240 Ala Glu Arg Asp Cys Ile Leu Phe Asp Thr Asp Ser Asn Tyr Ser Ser 245 250 255 Tyr Thr His Arg Val Leu Glu Gln Asp Arg Asn Arg Ala Asp Gln Ser 260 265 270 Leu Phe Ser Ile Asp Leu Glu Tyr Val His Asp Leu Glu Leu Ile Ala 275 280 285 Leu Gly Tyr Ser Asp Glu Asp Asp Glu Asp Leu Asp Asn Phe Phe 290 295 300 57256PRTCereal yellow dwarf virus 57Met Phe Ile Ala Gln Pro Cys Gly Arg Val Leu Val Phe Asp Val Ala 1 5 10 15 Ser Arg Thr Pro Ser Phe Phe Thr Arg Tyr Ser Val Glu Leu Ser Leu 20 25 30 Arg Val Leu Asp Pro Phe Phe Thr Arg Ala Val Thr Asp Phe Arg Tyr 35 40 45 Thr Gln Asn Glu Ile Asp Leu Phe Cys Val Ser Leu Gly Phe Leu Leu 50 55 60 Pro Ile Leu Leu Thr Gly Glu Ser Tyr Ser Trp Arg Gly His Leu Asn 65 70 75 80 Leu Pro Leu Ser Tyr Thr Glu Leu Leu Val Arg Trp Gly Leu Ala Val 85 90 95 Gly Tyr Phe Pro Thr Phe Ser Thr Asp Gly Asp Ile Arg Gln Asn Pro 100 105 110 Glu Leu Arg Ile Asp Leu Ser Thr Met Ser Thr Arg Ser Phe Tyr Glu 115 120 125 Gln Phe Leu Leu Arg Tyr Asn Thr Ser Gly Leu Ala Lys Ala Ile Val 130 135 140 Gly Gln Gln Glu Cys Phe Gln Ser Gly Met Glu Ser Phe Lys Arg Phe 145 150 155 160 Leu His Tyr Arg Leu Thr Cys Phe Glu Ser Cys Leu Pro Arg Pro Arg 165 170 175 Trp Glu Ser Pro Leu Ala Pro Gly Pro Tyr Leu Asp Arg Ala Phe Glu 180 185 190 Ala Thr Leu Leu Gly Arg Met Val Gly His Asn Gln Leu Leu Phe Thr 195 200 205 Gly Leu Ser Ser Asp Ile Thr Arg Tyr Tyr Asn Glu Leu Val Val Glu 210 215 220 Gly Val Pro Val Ala Phe Trp Asp Ala Ala Gly Ile Thr Leu His His 225 230 235 240 Ala Gly Glu Glu Tyr Phe Ser Asn Ser Tyr Ile Gln Lys Ile Leu Gln 245 250 255 58519DNATomato bushy stunt virus 58atggaacgag ctatacaagg aaacgacgct agggaacaag ctaacagtga acgttgggat 60ggaggatcag gaggtaccac ttctcccttc aaacttcctg acgaaagtcc gagttggact 120gagtggcggc tacataacga tgagacgaat tcgaatcaag ataatcccct tggtttcaag 180gaaagctggg gtttcgggaa agttgtattt aagagatatc tcagatacga caggacggaa 240gcttcactgc acagagtcct tggatcttgg acgggagatt cggttaacta tgcagcatct 300cgatttttcg gtttcgacca gatcggatgt acctatagta ttcggtttcg aggagttagt 360atcaccgttt ctggagggtc gcgaactctt cagcatctct gtgagatggc aattcggtct 420aagcaagaac tgctacagct tgccccaatc gaagtggaaa gtaatgtatc aagaggatgc 480cctgaaggta ctgagacctt cgaaaaagaa agcgagtaa 51959351DNATomato yellow leaf curl virus 59atgtgggatc cacttctaaa tgaatttcct gaatctgttc acggatttcg ttgtatgtta 60gctattaaat atttgcagtc cgttgaggaa acttacgagc ccaatacatt gggccacgat 120ttaattaggg atcttatatc tgttgtaagg gcccgtgact atgtcgaagc gaccaggcga 180tataatcatt tccacgcccg cctcgaaggt tcgccgaagg ctgaacttcg acagcccata 240cagcagccgt gctgctgtcc ccattgtcca aggcacaaac aagcgacgat catggacgta 300caggcccatg taccggaagc ccagaatata cagaatgtat cgaagccctg a 351601056DNATurnip crinkle virus 60atggaaaatg atcctagagt ccgaaagttc gcatccgagg gcgcccaatg ggcgataaag 60tggcagaaga agggctggtc atccctaacc agcagacaga aacagaccgc ccgcgcagcg 120atggggatca agctctcccc tgtggcgcaa cctgtgcaga aagtgactcg actgagtgct 180ccggtggctc tcgcctaccg cgaggtttcc acccagcctc gggtttctac tgccagggac 240ggcataacca gaagcggttc tgaactgatc acaaccctga agaagaacac tgacactgaa 300cctaagtaca ccacagctgt gcttaaccca agcgaacccg gaacattcaa ccaactcatc 360aaggaggcgg cccagtatga aaaataccga ttcacgtcac tcagatttag gtactctccc 420atgagccctt caaccaccgg gggcaaggtg gctctggcat tcgaccgaga cgctgccaaa 480cctccgccca acgacctcgc ttccctctac aacatagagg gttgtgtatc tagcgtgccc 540tggacagggt ttattttgac cgtcccaaca gattctactg accgctttgt ggcggatggt 600atcagcgatc caaagcttgt caatttcggc aagctcatca tggccaccta tggccaagga 660gccaatgatg ccgcccaact cggtgaagtg cgagtcgagt acaccgtgca gctcaagaac 720agaactggct caaccagcga cgcccagatt ggggacttcg cgggtgttaa ggacggaccc 780aggttggtct cgtggtccaa gaccaaggga acagctgggt gggagcacga ttgtcatttt 840ctcggaaccg gaaacttctc gttgacactg ttctacgaga aggcgcccgt ttcggggcta 900gaaaacgcag acgcctctga cttctcggtc ctgggagaag ccgcagcagg tagtgtccaa 960tgggctggag tgaaggtagc agaaagggga caaagcgtga aaatggtcac aactgaggag 1020cagccaaggg gaaaatggca agcactcagg atttag 105661912DNAPea mosaic virus 61atgcacggaa ttgagcagcc tcaactaccg ctagattacg ttcaccgttg cgcatcaacc 60tccttcttgc tcgcatcact agatggcctc ctttctgaag cccgtgaact ctcagggcct 120ctggctctca ttacttctag ctattactta cttgtttcta ttgccctctg ctgggcaatc 180cctggatcct tctggtatag gcctggctgc tggttgcagc cagtctcagg gcggaatctc 240atcttttgcg gccctaccga ggccttgcaa cgattccgtc tgtacgctgc cagacttggg 300ttggtcctgt cagagaactg cccaagacac ggccaatcag cagcaatcac ccttcaatca 360tactgggcac ttcctaacaa catctggatg gacatggccc aattggactt gctcaccttc 420tcaatgccaa ttgctaatac atttgcctac ttggcagatt gtgaagcaag atttcctcct 480attgttgaag gagtgggatc tgcttactat gtgccaacgc tgctcggact tactcaccaa 540gaccccaggc tttatcttgc gcttcgcagg agaaaccttg atcttagtgg cgaacctcat 600agagttcgtc ctggtgtcct ggagtctatg gctttgctct gttctagtgt acgtagcaca 660agccgttcca ggcaaattcc tcctttatat ggcagcgttt tgcaccacgt tttgggcctg 720gccgagagag actgcatcct ctttgatacg gatagtaact actcctctta cactcatcgg 780gttcttgaac aagaccggaa tcgggctgat cagtcattgt ttagcattga cttggaatat 840gttcatgacc tggagcttat tgccctgggt tactctgatg aagatgatga agatcttgat 900aacttcttct ag 91262771DNACereal yellow dwarf virus 62atgttcatcg cccaaccttg cgggcgagtt cttgtgttcg acgtcgcctc ccgcacgcca 60tcgttcttca ctcgttatag tgttgaactc tcgctccgtg ttctagaccc attcttcacg 120cgagcagtaa cagatttccg atacacccaa aatgaaatcg atttattttg tgtgtctctt 180ggctttctgt tgccaattct cctcacagga gaatcttact cttggcgcgg tcacctcaac 240ctcccccttt cttacaccga attacttgtt cgatgggggc tcgcagtggg gtacttccct 300accttctcca ctgatggtga cattcgacag aacccagaac tccgcatcga cctgtccacc 360atgtcaaccc gctctttcta cgagcagttc ctactcagat ataacacgag tgggttggca 420aaagctatcg tcggacagca agagtgcttt caaagcggca tggagtcttt taaaagattc 480ctacactacc gcctcacgtg ctttgaaagc tgccttccac gacctcgttg ggaaagtcct 540ttggctcctg gtccttatct ggacagggct tttgaggcaa ctcttctcgg ccgtatggtc 600ggtcataacc aactactctt taccggtttg tcttctgata tcactaggta ttataacgag 660ttggttgtgg aaggcgtgcc ggtggctttt tgggacgctg ccggcattac tttgcatcac 720gctggtgaag aatatttttc gaattcttac attcaaaaga ttcttcaatg a 77163389PRTArabidopsis thaliana 63Met Val Ile Ala Ala Ala Val Ile Val Pro Leu Gly Leu Leu Phe Phe 1 5 10 15 Ile Ser Gly Leu Ala Val Asn Leu Phe Gln Ala Val Cys Tyr Val Leu 20 25 30 Ile Arg Pro Leu Ser Lys Asn Thr Tyr Arg Lys Ile Asn Arg Val Val 35 40 45 Ala Glu Thr Leu Trp Leu Glu Leu Val Trp Ile Val Asp Trp Trp Ala 50 55 60 Gly Val Lys Ile Gln Val Phe Ala Asp Asn Glu Thr Phe Asn Arg Met 65 70 75 80 Gly Lys Glu His Ala Leu Val Val Cys Asn His Arg Ser Asp Ile Asp 85 90 95 Trp Leu Val Gly Trp Ile Leu Ala Gln Arg Ser Gly Cys Leu Gly Ser 100 105 110 Ala Leu Ala Val Met Lys Lys Ser Ser Lys Phe Leu Pro Val Ile Gly 115 120 125 Trp Ser Met Trp Phe Ser Glu Tyr Leu Phe Leu Glu Arg Asn Trp Ala 130 135 140 Lys Asp Glu Ser Thr Leu Lys Ser Gly Leu Gln Arg Leu Ser Asp Phe 145 150 155 160 Pro Arg Pro Phe Trp Leu Ala Leu Phe Val Glu Gly Thr Arg Phe Thr 165 170 175 Glu Ala Lys Leu Lys Ala Ala Gln Glu Tyr Ala Ala Ser Ser Glu Leu 180 185 190 Pro Ile Pro Arg Asn Val Leu Ile Pro Arg Thr Lys Gly Phe Val Ser 195 200 205 Ala Val Ser Asn Met Arg Ser Phe Val Pro Ala Ile Tyr Asp Met Thr 210 215 220 Val Thr Ile Pro Lys Thr Ser Pro Pro Pro Thr Met Leu Arg Leu Phe 225 230 235 240 Lys Gly Gln Pro Ser Val Val His Val His Ile Lys Cys His Ser Met 245 250 255 Lys Asp Leu Pro Glu Ser Asp Asp Ala Ile Ala Gln Trp Cys Arg Asp 260 265 270 Gln Phe Val Ala Lys Asp Ala Leu Leu Asp Lys His Ile Ala Ala Asp 275 280 285 Thr Phe Pro Gly Gln Gln Glu Gln Asn Ile Gly Arg Pro Ile Lys Ser 290 295 300 Leu Ala Val Val Leu Ser Trp Ala Cys Val Leu Thr Leu Gly Ala Ile 305 310 315 320 Lys Phe Leu His Trp Ala Gln Leu Phe Ser Ser Trp Lys Gly Ile Thr 325 330 335 Ile Ser Ala Leu Gly Leu Gly Ile Ile Thr Leu Cys Met Gln Ile Leu 340 345 350 Ile Arg Ser Ser Gln Ser Glu Arg Ser Thr Pro Ala Lys Val Val Pro 355 360 365 Ala Lys Pro Lys Asp Asn His His Pro Glu Ser Ser Ser Gln Thr Glu 370 375 380 Thr Glu Lys Glu Lys 385 64281PRTLimnanthes alba 64Met Ala Lys Thr Arg Thr Ser Ser Leu Arg Asn Arg Arg Gln Leu Lys 1 5 10 15 Thr Ala Val Ala Ala Thr Ala Asp Asp Asp Lys Asp Gly Ile Phe Met 20 25 30 Val Leu Leu Ser Cys Phe Lys Ile Phe Val Cys Phe Ala Ile Val Leu 35 40 45 Ile Thr Ala Val Ala Trp Gly Leu Ile Met Val Leu Leu Leu Pro Trp 50 55 60 Pro Tyr Met Arg Ile Arg Leu Gly Asn Leu Tyr Gly His Ile Ile Gly 65 70 75 80 Gly Leu Val Ile Trp Leu Tyr Gly Ile Pro Ile Glu Ile Gln Gly Ser 85 90 95 Glu His Thr Lys Lys Arg Ala Ile Tyr Ile Ser Asn His Ala Ser Pro 100 105 110 Ile Asp Ala Phe Phe Val Met Trp Leu Ala Pro Ile Gly Thr Val Gly 115 120 125 Val Ala Lys Lys Glu Val Ile Trp Tyr Pro Leu Leu Gly Gln Leu Tyr 130 135 140 Thr Leu Ala His His Ile Arg Ile Asp Arg Ser Asn Pro Ala Ala Ala 145 150 155 160 Ile Gln Ser Met Lys Glu Ala Val Arg Val Ile Thr Glu Lys Asn Leu 165 170 175 Ser Leu Ile Met Phe Pro Glu Gly Thr Arg Ser Gly Asp Gly Arg Leu 180 185 190 Leu Pro Phe Lys Lys Gly Phe Val His Leu Ala Leu Gln Ser His Leu 195 200 205 Pro Ile Val Pro Met Ile Leu Thr Gly Thr His Leu Ala Trp Arg Lys 210 215 220 Gly Thr Phe Arg Val Arg Pro Val Pro Ile Thr Val Lys Tyr Leu Pro 225 230 235 240 Pro Ile Asn Thr Asp Asp Trp Thr Val Asp Lys Ile Asp Asp Tyr Val 245 250 255 Lys Met Ile His Asp Ile Tyr Val Arg Asn Leu Pro Ala Ser Gln Lys 260 265 270 Pro Leu Gly Ser Thr Asn Arg Ser Lys 275 280 65303PRTSaccharomyces cerevisiae 65Met Ser Val Ile Gly Arg Phe Leu Tyr Tyr Leu Arg Ser Val Leu Val 1 5 10 15 Val Leu Ala Leu Ala Gly Cys Gly Phe Tyr Gly Val Ile Ala Ser Ile 20 25 30 Leu Cys Thr Leu Ile Gly Lys Gln His Leu Ala Gln Trp Ile Thr Ala 35 40 45 Arg Cys Phe Tyr His Val Met Lys Leu Met Leu Gly Leu Asp Val Lys 50 55 60 Val Val Gly Glu Glu Asn Leu Ala Lys Lys Pro Tyr Ile Met Ile Ala 65 70 75 80 Asn His Gln Ser Thr Leu Asp Ile Phe Met Leu Gly Arg Ile Phe Pro 85 90 95 Pro Gly Cys Thr Val Thr Ala Lys Lys Ser Leu Lys Tyr Val Pro Phe 100 105 110 Leu Gly Trp Phe Met Ala Leu Ser Gly Thr Tyr Phe Leu Asp Arg Ser 115 120 125 Lys Arg Gln Glu Ala Ile Asp Thr Leu Asn Lys Gly Leu Glu Asn Val 130 135 140 Lys Lys Asn Lys Arg Ala Leu Trp Val Phe Pro Glu Gly Thr Arg Ser 145 150 155

160 Tyr Thr Ser Glu Leu Thr Met Leu Pro Phe Lys Lys Gly Ala Phe His 165 170 175 Leu Ala Gln Gln Gly Lys Ile Pro Ile Val Pro Val Val Val Ser Asn 180 185 190 Thr Ser Thr Leu Val Ser Pro Lys Tyr Gly Val Phe Asn Arg Gly Cys 195 200 205 Met Ile Val Arg Ile Leu Lys Pro Ile Ser Thr Glu Asn Leu Thr Lys 210 215 220 Asp Lys Ile Gly Glu Phe Ala Glu Lys Val Arg Asp Gln Met Val Asp 225 230 235 240 Thr Leu Lys Glu Ile Gly Tyr Ser Pro Ala Ile Asn Asp Thr Thr Leu 245 250 255 Pro Pro Gln Ala Ile Glu Tyr Ala Ala Leu Gln His Asp Lys Lys Val 260 265 270 Asn Lys Lys Ile Lys Asn Glu Pro Val Pro Ser Val Ser Ile Ser Asn 275 280 285 Asp Val Asn Thr His Asn Glu Gly Ser Ser Val Lys Lys Met His 290 295 300 66373PRTMicromonas pusilla 66Met Thr Pro Tyr Gln Trp Phe Asn Val Val Ser Ser Leu Gly Tyr Val 1 5 10 15 Leu Phe Thr Ala Thr Thr Ser Thr Val Thr Met Leu Val Pro Ala Ile 20 25 30 Ile Leu Leu Arg Pro Val Ser Ala Asn Leu Tyr Ala Arg Cys Thr Ser 35 40 45 Trp Ile Phe Ala Cys Trp Trp Thr Ser Cys Leu Phe Ile Thr Glu Arg 50 55 60 Leu Asn Gly Val Lys Val Arg Val Thr Gly Asp Ala Leu Pro Leu Asn 65 70 75 80 Ala Pro Leu Leu Ile Met Ser Asn His Lys Cys Asn Leu Asp Trp Met 85 90 95 Phe Leu Trp Ser Ser Ala Ile Arg Thr Gly Ser Met Phe His Val Gly 100 105 110 Val Phe Lys Ala Val Ala Lys Ser Glu Ile Arg Val Ile Pro Ile Phe 115 120 125 Gly Trp Gly Cys Lys Leu Asn Gly Phe Ala Tyr Val Arg Arg Arg Trp 130 135 140 Ser Ser Asp Ala Ser His Leu Thr Ser Trp Ile Gln Ser Gln Ile Arg 145 150 155 160 Arg Arg Leu Asn Ala Asn Trp Thr Leu Ile Phe Pro Glu Gly Thr Arg 165 170 175 Tyr Thr Asp Arg Asn Lys Glu Arg Ser Asp Leu Ser Cys Ala Lys Asp 180 185 190 Gly Leu Glu Pro Met Ala Gly Glu Ile Leu Arg Pro Arg Thr Lys Gly 195 200 205 Leu Ala Leu Leu Leu Arg Glu Ser Ala Lys Gly Gly Gly Tyr Tyr Arg 210 215 220 Lys Ile Val Asp Met Thr Ile Gln Tyr Thr Asp Ala Asp Gly Lys Pro 225 230 235 240 Leu Lys Gly Ala Ala Leu Gly Thr Arg Cys Phe Gly Gln Leu Ala Lys 245 250 255 Gly Gln Leu Pro Val Ala Thr Cys His Val His Phe Asp Val Phe Ser 260 265 270 His Lys Asp Val Pro Ala Gly Glu Asp Glu Asp Glu Val Glu Ala Trp 275 280 285 Val Trp Lys Arg Trp Arg Lys Lys Ala Asn Met Leu Glu Ala Cys Ala 290 295 300 Ser Ala Gly Gln Phe Glu Gly Val Arg Glu Trp Ser Thr Ser Gly Thr 305 310 315 320 Ala Val Pro Leu Lys Thr Gln Thr Ala Leu Arg Cys Phe Phe Val Leu 325 330 335 Gln Gly Leu Val Cys Val Gly Val Ala Cys Ser Ser Thr Ala Phe Leu 340 345 350 Ala Tyr Val Ala Cys Ala Ala Val Gly Ala Ala Val Ile Ala Gln Thr 355 360 365 Asp Pro Ala Trp Trp 370 67314PRTMortierella alpina 67Met Ser Ile Gly Ser Ser Asn Pro Val Leu Leu Ala Ala Ile Pro Phe 1 5 10 15 Val Tyr Leu Phe Val Leu Pro Arg Val Leu Ala Phe Leu Pro Gln Lys 20 25 30 Ala Gln Phe Leu Ala Lys Cys Ile Val Val Leu Ile Ala Thr Leu Ile 35 40 45 Met Ser Val Ala Gly Cys Phe Ile Ser Ile Val Cys Ala Leu Leu Asp 50 55 60 Lys Arg Tyr Val Ile Asn Tyr Val Val Ser Arg Leu Phe Ser Phe Leu 65 70 75 80 Ala Ala Arg Pro Cys Gly Val Thr Tyr Lys Ile Val Gly Glu Glu His 85 90 95 Leu Asp Lys Tyr Pro Ala Ile Val Val Cys Asn His Gln Ser Ser Met 100 105 110 Asp Met Met Val Leu Gly Arg Val Phe Pro Lys His Cys Val Val Met 115 120 125 Ala Lys Lys Glu Leu Leu Tyr Phe Pro Phe Leu Gly Met Phe Met Lys 130 135 140 Leu Ser Asn Ala Ile Phe Ile Asp Arg Lys Asn His Lys Lys Ala Ile 145 150 155 160 Glu Ser Thr Thr Gln Ala Val Ala Asp Met Lys Lys His Asn Ser Gly 165 170 175 Ile Trp Ile Phe Pro Glu Gly Thr Arg Ser Arg Leu Asp Lys Ala Asp 180 185 190 Leu Leu Pro Phe Lys Lys Gly Ala Phe His Leu Ala Ile Gln Ala Gln 195 200 205 Leu Pro Ile Leu Pro Ile Ile Ser Gln Gly Tyr Ser His Ile Tyr Asp 210 215 220 Ser Ser Lys Arg Tyr Phe Pro Gly Gly Glu Leu Glu Ile Arg Val Leu 225 230 235 240 Glu Pro Ile Pro Thr Thr Gly Leu Thr Thr Asp Asp Val Asn Asp Leu 245 250 255 Met Asp Lys Thr Arg Asn Leu Met Leu Lys His Leu Lys Glu Met Asp 260 265 270 Ser Gln Tyr Ser Ser Ser Thr Ala Glu Asn Gly Ser Thr His Ile Asp 275 280 285 Ala Asp Ile Ala Lys Ser Thr Ala Thr Ser Ile Gly Asn Thr Asp Asp 290 295 300 Ala Ile Thr Lys Arg Arg Thr Pro Lys Glu 305 310 68391PRTBraccisa napus 68Met Ala Met Ala Ala Ala Ala Val Ile Val Pro Leu Gly Ile Leu Phe 1 5 10 15 Phe Ile Ser Gly Leu Val Val Asn Leu Leu Gln Ala Val Cys Tyr Val 20 25 30 Leu Ile Arg Pro Leu Ser Lys Asn Thr Tyr Arg Lys Ile Asn Arg Val 35 40 45 Val Ala Glu Thr Leu Trp Leu Glu Leu Val Trp Ile Val Asp Trp Trp 50 55 60 Ala Gly Val Lys Ile Gln Val Phe Ala Asp Asp Glu Thr Phe Asn Arg 65 70 75 80 Met Gly Lys Glu His Ala Leu Val Val Cys Asn His Arg Ser Asp Ile 85 90 95 Asp Trp Leu Val Gly Trp Ile Leu Ala Gln Arg Ser Gly Cys Leu Gly 100 105 110 Ser Ala Leu Ala Val Met Lys Lys Ser Ser Lys Phe Leu Pro Val Ile 115 120 125 Gly Trp Ser Met Trp Phe Ser Glu Tyr Leu Phe Leu Glu Arg Asn Trp 130 135 140 Ala Lys Asp Glu Ser Thr Leu Lys Ser Gly Leu Gln Arg Leu Asn Asp 145 150 155 160 Phe Pro Arg Pro Phe Trp Leu Ala Leu Phe Val Glu Gly Thr Arg Phe 165 170 175 Thr Glu Ala Lys Leu Lys Ala Ala Gln Glu Tyr Ala Ala Ser Ser Gln 180 185 190 Leu Pro Val Pro Arg Asn Val Leu Ile Pro Arg Thr Lys Gly Phe Val 195 200 205 Ser Ala Val Ser Asn Met Arg Ser Phe Val Pro Ala Ile Tyr Asp Met 210 215 220 Thr Val Ala Ile Pro Lys Thr Ser Pro Pro Pro Thr Met Leu Arg Leu 225 230 235 240 Phe Lys Gly Gln Pro Ser Val Val His Val His Ile Lys Cys His Ser 245 250 255 Met Lys Asp Leu Pro Glu Ser Asp Asp Ala Ile Ala Gln Trp Cys Arg 260 265 270 Asp Gln Phe Val Ala Lys Asp Ala Leu Leu Asp Lys His Ile Ala Ala 275 280 285 Asp Thr Phe Pro Gly Gln Lys Glu His Asn Ile Gly Arg Pro Ile Lys 290 295 300 Ser Leu Ala Val Val Val Ser Trp Ala Cys Leu Leu Thr Leu Gly Ala 305 310 315 320 Met Lys Phe Leu His Trp Ser Asn Leu Phe Ser Ser Leu Lys Gly Ile 325 330 335 Ala Leu Ser Ala Leu Gly Leu Gly Ile Ile Thr Leu Cys Met Gln Ile 340 345 350 Leu Ile Arg Ser Ser Gln Ser Glu Arg Ser Thr Pro Ala Lys Val Ala 355 360 365 Pro Ala Lys Pro Lys Asp Lys His Gln Ser Gly Ser Ser Ser Gln Thr 370 375 380 Glu Val Glu Glu Lys Gln Lys 385 390 69390PRTBraccisa napus 69Met Ala Met Ala Ala Ala Val Ile Val Pro Leu Gly Ile Leu Phe Phe 1 5 10 15 Ile Ser Gly Leu Val Val Asn Leu Leu Gln Ala Ile Cys Tyr Val Leu 20 25 30 Ile Arg Pro Leu Ser Lys Asn Thr Tyr Arg Lys Ile Asn Arg Val Val 35 40 45 Ala Glu Thr Leu Trp Leu Glu Leu Val Trp Ile Val Asp Trp Trp Ala 50 55 60 Gly Val Lys Ile Gln Val Phe Ala Asp Asn Glu Thr Phe Asn Arg Met 65 70 75 80 Gly Lys Glu His Ala Leu Val Val Cys Asn His Arg Ser Asp Ile Asp 85 90 95 Trp Leu Val Gly Trp Ile Leu Ala Gln Arg Ser Gly Cys Leu Gly Ser 100 105 110 Ala Leu Ala Val Met Lys Lys Ser Ser Lys Phe Leu Pro Val Ile Gly 115 120 125 Trp Ser Met Trp Phe Ser Glu Tyr Leu Phe Leu Glu Arg Asn Trp Ala 130 135 140 Lys Asp Glu Ser Thr Leu Lys Ser Gly Leu Gln Arg Leu Asn Asp Phe 145 150 155 160 Pro Arg Pro Phe Trp Leu Ala Leu Phe Val Glu Gly Thr Arg Phe Thr 165 170 175 Glu Ala Lys Leu Lys Ala Ala Gln Glu Tyr Ala Ala Ser Ser Glu Leu 180 185 190 Pro Val Pro Arg Asn Val Leu Ile Pro Arg Thr Lys Gly Phe Val Ser 195 200 205 Ala Val Ser Asn Met Arg Ser Phe Val Pro Ala Ile Tyr Asp Met Thr 210 215 220 Val Ala Ile Pro Lys Thr Ser Pro Pro Pro Thr Met Leu Arg Leu Phe 225 230 235 240 Lys Gly Gln Pro Ser Val Val His Val His Ile Lys Cys His Ser Met 245 250 255 Lys Asp Leu Pro Glu Ser Asp Asp Ala Ile Ala Gln Trp Cys Arg Asp 260 265 270 Gln Phe Val Ala Lys Asp Ala Leu Leu Asp Lys His Ile Ala Ala Asp 275 280 285 Thr Phe Pro Gly Gln Gln Glu Gln Asn Ile Gly Arg Pro Ile Lys Ser 290 295 300 Leu Ala Val Val Leu Ser Trp Ser Cys Leu Leu Ile Leu Gly Ala Met 305 310 315 320 Lys Phe Leu His Trp Ser Asn Leu Phe Ser Ser Trp Lys Gly Ile Ala 325 330 335 Phe Ser Ala Leu Gly Leu Gly Ile Ile Thr Leu Cys Met Gln Ile Leu 340 345 350 Ile Arg Ser Ser Gln Ser Glu Arg Ser Thr Pro Ala Lys Val Val Pro 355 360 365 Ala Lys Pro Lys Asp Asn His Asn Asp Ser Gly Ser Ser Ser Gln Thr 370 375 380 Glu Val Glu Lys Gln Lys 385 390 70361PRTPhytophthora infestans 70Met Ala Thr Lys Glu Ala Tyr Val Phe Pro Thr Leu Thr Glu Ile Lys 1 5 10 15 Arg Ser Leu Pro Lys Asp Cys Phe Glu Ala Ser Val Pro Leu Ser Leu 20 25 30 Tyr Tyr Thr Val Arg Cys Leu Val Ile Ala Val Ala Leu Thr Phe Gly 35 40 45 Leu Asn Tyr Ala Arg Ala Leu Pro Glu Val Glu Ser Phe Trp Ala Leu 50 55 60 Asp Ala Ala Leu Cys Thr Gly Tyr Ile Leu Leu Gln Gly Ile Val Phe 65 70 75 80 Trp Gly Phe Phe Thr Val Gly His Asp Ala Gly His Gly Ala Phe Ser 85 90 95 Arg Tyr His Leu Leu Asn Phe Val Val Gly Thr Phe Met His Ser Leu 100 105 110 Ile Leu Thr Pro Phe Glu Ser Trp Lys Leu Thr His Arg His His His 115 120 125 Lys Asn Thr Gly Asn Ile Asp Arg Asp Glu Val Phe Tyr Pro Gln Arg 130 135 140 Lys Ala Asp Asp His Pro Leu Ser Arg Asn Leu Ile Leu Ala Leu Gly 145 150 155 160 Ala Ala Trp Leu Ala Tyr Leu Val Glu Gly Phe Pro Pro Arg Lys Val 165 170 175 Asn His Phe Asn Pro Phe Glu Pro Leu Phe Val Arg Gln Val Ser Ala 180 185 190 Val Val Ile Ser Leu Leu Ala His Phe Phe Val Ala Gly Leu Ser Ile 195 200 205 Tyr Leu Ser Leu Gln Leu Gly Leu Lys Thr Met Ala Ile Tyr Tyr Tyr 210 215 220 Gly Pro Val Phe Val Phe Gly Ser Met Leu Val Ile Thr Thr Phe Leu 225 230 235 240 His His Asn Asp Glu Glu Thr Pro Trp Tyr Ala Asp Ser Glu Trp Thr 245 250 255 Tyr Val Lys Gly Asn Leu Ser Ser Val Asp Arg Ser Tyr Gly Ala Leu 260 265 270 Ile Asp Asn Leu Ser His Asn Ile Gly Thr His Gln Ile His His Leu 275 280 285 Phe Pro Ile Ile Pro His Tyr Lys Leu Lys Lys Ala Thr Ala Ala Phe 290 295 300 His Gln Ala Phe Pro Glu Leu Val Arg Lys Ser Asp Glu Pro Ile Ile 305 310 315 320 Lys Ala Phe Phe Arg Val Gly Arg Leu Tyr Ala Asn Tyr Gly Val Val 325 330 335 Asp Gln Glu Ala Lys Leu Phe Thr Leu Lys Glu Ala Lys Ala Ala Thr 340 345 350 Glu Ala Ala Ala Lys Thr Lys Ser Thr 355 360 71418PRTThalassiosira pseudonana 71Met Tyr Arg Leu Thr Ser Thr Phe Leu Ile Ala Leu Ala Phe Ser Ser 1 5 10 15 Ser Ile Asn Ala Phe Ser Pro Gln Arg Pro Pro Arg Thr Ile Thr Lys 20 25 30 Ser Lys Val Gln Ser Thr Val Leu Pro Ile Pro Thr Lys Asp Asp Leu 35 40 45 Asn Phe Leu Gln Pro Gln Leu Asp Glu Asn Asp Leu Tyr Leu Asp Asp 50 55 60 Val Asn Thr Pro Pro Arg Ala Gly Thr Ile Met Lys Met Leu Pro Lys 65 70 75 80 Glu Thr Phe Asn Ile Asp Thr Ala Thr Ser Leu Gly Tyr Phe Gly Met 85 90 95 Asp Met Ala Ala Val Val Ser Ser Met Thr Leu Leu Asn Ala Ile Val 100 105 110 Thr Ser Asp Gln Tyr His Ala Leu Pro Leu Pro Leu Gln Ala Ala Thr 115 120 125 Val Ile Pro Phe Gln Leu Leu Ala Gly Phe Ala Met Trp Cys Met Trp 130 135 140 Cys Ile Gly His Asp Ala Gly His Ser Thr Val Ser Lys Thr Lys Trp 145 150 155 160 Ile Asn Arg Val Val Gly Glu Val Ala His Ser Val Val Cys Leu Thr 165 170 175 Pro Phe Val Pro Trp Gln Met Ser His Arg Lys His His Leu Asn His 180 185 190 Asn His Ile Glu Lys Asp Tyr Ser His Lys Trp Tyr Ser Arg Asp Glu 195 200 205 Phe Asp Asp Ile Pro Gln Leu Tyr Lys Thr Phe Gly Tyr Asn Pro Arg 210 215 220 Met Met Gln Leu Pro Phe Leu Tyr Phe Met Tyr Leu Ala Leu Gly Ile 225 230 235 240 Pro Asp Gly Gly His Val Val Phe Tyr Gly Arg Met Trp Glu Gly Val 245 250 255 Ser Leu Gln Lys Lys Phe Asp Ala Ala Ile Ser Val Ala Val Ser Cys 260 265 270 Ala Thr Ala Gly Ser Leu Trp Met Asn Met Gly Thr Ala Asp Phe Thr 275 280 285 Val Val Cys Met Val Pro Trp Leu Val Leu Ser Trp Trp Leu Phe Met 290 295 300 Val Thr Tyr Leu Gln His

His Ser Glu Asp Gly Lys Leu Tyr Thr Asp 305 310 315 320 Glu Thr Phe Thr Phe Glu Lys Gly Ala Phe Glu Thr Val Asp Arg Ser 325 330 335 Tyr Gly Lys Leu Ile Asn Arg Met Ser His His Met Met Asp Gly His 340 345 350 Val Val His His Leu Phe Phe Glu Arg Val Pro His Tyr Arg Leu Glu 355 360 365 Ala Ala Thr Glu Ala Leu Val Lys Gly Met Asp Glu Thr Gly Gln Lys 370 375 380 His Leu Tyr Lys Tyr Ile Asp Thr Pro Asp Phe Asn Ala Glu Ile Val 385 390 395 400 Asn Gly Phe Arg Asp Asn Trp Phe Leu Val Glu Glu Glu Asn Ile Lys 405 410 415 Arg Glu 72363PRTPythium irregulare 72Met Ala Ser Thr Ser Ala Ala Gln Asp Ala Ala Pro Tyr Glu Phe Pro 1 5 10 15 Ser Leu Thr Glu Ile Lys Arg Ala Leu Pro Ser Glu Cys Phe Glu Ala 20 25 30 Ser Val Pro Leu Ser Leu Tyr Tyr Thr Ala Arg Ser Leu Ala Leu Ala 35 40 45 Gly Ser Leu Ala Val Ala Leu Ser Tyr Ala Arg Ala Leu Pro Leu Val 50 55 60 Gln Ala Asn Ala Leu Leu Asp Ala Thr Leu Cys Thr Gly Tyr Val Leu 65 70 75 80 Leu Gln Gly Ile Val Phe Trp Gly Phe Phe Thr Val Gly His Asp Cys 85 90 95 Gly His Gly Ala Phe Ser Arg Ser His Val Leu Asn Phe Ser Val Gly 100 105 110 Thr Leu Met His Ser Ile Ile Leu Thr Pro Phe Glu Ser Trp Lys Leu 115 120 125 Ser His Arg His His His Lys Asn Thr Gly Asn Ile Asp Lys Asp Glu 130 135 140 Ile Phe Tyr Pro Gln Arg Glu Ala Asp Ser His Pro Val Ser Arg His 145 150 155 160 Leu Val Met Ser Leu Gly Ser Ala Trp Phe Ala Tyr Leu Phe Ala Gly 165 170 175 Phe Pro Pro Arg Thr Met Asn His Phe Asn Pro Trp Glu Ala Met Tyr 180 185 190 Val Arg Arg Val Ala Ala Val Ile Ile Ser Leu Gly Val Leu Phe Ala 195 200 205 Phe Ala Gly Leu Tyr Ser Tyr Leu Thr Phe Val Leu Gly Phe Thr Thr 210 215 220 Met Ala Ile Tyr Tyr Phe Gly Pro Leu Phe Ile Phe Ala Thr Met Leu 225 230 235 240 Val Val Thr Thr Phe Leu His His Asn Asp Glu Glu Thr Pro Trp Tyr 245 250 255 Ala Asp Ser Glu Trp Thr Tyr Val Lys Gly Asn Leu Ser Ser Val Asp 260 265 270 Arg Ser Tyr Gly Ala Leu Ile Asp Asn Leu Ser His Asn Ile Gly Thr 275 280 285 His Gln Ile His His Leu Phe Pro Ile Ile Pro His Tyr Lys Leu Asn 290 295 300 Asp Ala Thr Ala Ala Phe Ala Lys Ala Phe Pro Glu Leu Val Arg Lys 305 310 315 320 Asn Ala Ala Pro Ile Ile Pro Thr Phe Phe Arg Met Ala Ala Met Tyr 325 330 335 Ala Lys Tyr Gly Val Val Asp Thr Asp Ala Lys Thr Phe Thr Leu Lys 340 345 350 Glu Ala Lys Ala Ala Ala Lys Thr Lys Ser Ser 355 360

Claims

1-85. (canceled)

86. Extracted lipid from Brassica or Arabidopsis seeds, whose total fatty acid content comprises (a) a total monounsaturated fatty acid content which comprises oleic acid, (b) a total saturated fatty acid content which comprises palmitic acid and, if present, myristic acid (C14:0), (c) a total ω6 fatty acid content which comprises linoleic acid (LA), (d) a ω3 fatty acid content, (e) less than 1% myristic acid (C14:0), and (f) less than 4% eicosatrienoic acid (ETrA), wherein (i) a level of DNA between 7% and 20% is present in the total fatty acid content of the extracted lipid, wherein at least 70% of the DHA is esterified at the sn-1 or sn-3 position of triacylglycerol (TAG), (ii) a level of palmitic acid between 2% and 16% is present in the total fatty acid content of the extracted lipid, (iii) a level of linoleic acid (LA) between 4% and 35% is present in the total fatty acid content of the extracted lipid, (iv) the total saturated fatty acid content of the extracted lipid is between 4% and 25% of the total fatty acid content, (v) the ratio of the total ω6 fatty acid content to the ω3 fatty acid content in total of the extracted lipid is between 0.1 and 3.0, and (vi) at least 70% of the total fatty acid content of the extracted lipid is esterified in the form of TAG.

87. The extracted lipid of claim 86 which has one or more or all of the following features i) a level of palmitic acid between 2% and 15% is present in the total fatty acid content of the extracted lipid, ii) a level of eicosatrienoic acid (ETrA) between 0.05% and about 2% is present in the total fatty acid content of the extracted lipid, iii) the extracted lipid has a level of SDA of less than about 0.1%, EPA of less than about 0.1%, and ETA of less than about 0.1% in its total fatty acid content, iv) the total monounsaturated fatty acid content in the extracted lipid is between about 4% and about 35% of the total fatty acid content, v) the extracted lipid comprises new ω6 fatty acids in the total ω6 fatty acid content, wherein the level of new ω6 fatty acids in the extracted lipid is less than about 10% of the total fatty acid content, vi) the ω3 fatty acid content in total in the extracted lipid is between 36% and about 65% of the total fatty acid content, vii) the ratio of the total ω6 fatty acid content to the ω3 fatty acid content in total in the extracted lipid is between about 0.1 and about 0.5, viii) the extracted lipid comprises new ω3 fatty acids in the ω3 fatty acid content in total and new ω6 fatty acids in the total ω6 fatty acid content, wherein the ratio of the new ω6 fatty acids to the new ω3 fatty acids in the fatty acid content of the extracted lipid is between about 0.1 and about 1, ix) the extracted lipid has a fatty acid composition based on an efficiency of conversion of oleic acid to DHA of at least about 10%, x) the extracted lipid has a fatty acid composition based on an efficiency of conversion of LA to DHA of at least about 15%, xi) the extracted lipid has a fatty acid composition based on an efficiency of conversion of ALA to DHA of at least about 17%, xii) the total fatty acid content in the extracted lipid has less than 1% C20:1, xiii) at least about 80% of the total fatty acid content of the extracted lipid is esterified in the form of TAG, xiv) the extracted lipid comprises diacylglycerol (DAG), and xv) the extracted lipid comprises less than about 10% free (non-esterified) fatty acids and/or phospholipid.

88. The extracted lipid of claim 86 which is in the form of an oil, wherein the oil comprises one or more sterols.

89. The extracted lipid of claim 88, wherein the oil comprises less than 10 mg of sterols/g of oil.

90. The extracted lipid of claim 88, wherein the oil comprises one or more or all of campesterol, Δ5-stigmasterol, eburicol, β-sitosterol, Δ5-avenasterol, Δ7-stigmasterol and Δ7-avenasterol.

91. The extracted lipid of claim 86, which is from canola seed.

92. The extracted lipid of claim 86, wherein a level of linoleic acid between 4% and about 20% is present in the total fatty acid content of the extracted lipid.

93. The extracted lipid of claim 86, wherein a level of α-linolenic acid (ALA) between about 10% and about 35% is present in the total fatty acid content of the extracted lipid.

94. The extracted lipid of claim 86, wherein a level of α-linolenic acid (ALA) between about 2% and about 16% is present in the total fatty acid content of the extracted lipid.

95. The extracted lipid of claim 86, wherein a level of γ-linolenic acid (GLA) less than 4% is present in the total fatty acid content of the extracted lipid.

96. The extracted lipid of claim 86, wherein a level of stearidonic acid (SDA) between 0.05% and about 7% is present in the total fatty acid content of the extracted lipid.

97. The extracted lipid of claim 86, wherein a level of eicosatetraenoic acid (ETA) between 0.05% and about 4% is present in the total fatty acid content of the extracted lipid.

98. The extracted lipid of claim 86, wherein a level of eicosapentaenoic acid (EPA) less than 4% is present in the total fatty acid content of the extracted lipid.

99. The extracted lipid of claim 86, wherein a level of docosapentaenoic acid (DPA) between 0.05% and 8% is present in the total fatty acid content of the extracted lipid.

100. The extracted lipid of claim 86, wherein a level of DHA between about 10% and 20% is present in the total fatty acid content of the extracted lipid.

101. The extracted lipid of claim 86, wherein a level of DHA between about 11% and 20% is present in the total fatty acid content of the extracted lipid.

102. The extracted lipid of claim 86, wherein the extracted lipid comprises less than about 0.1% ω6-docosapentaenoic acid (22:5.sup.Δ4, 7, 10, 13, 16) in its total fatty acid content.

103. The extracted lipid of claim 86, wherein the total saturated fatty acid content in the extracted lipid is between about 4% and about 20% of the total fatty acid content.

104. The extracted lipid of claim 86, wherein the total monounsaturated fatty acid content in the extracted lipid is between about 4% and about 35% of the total fatty acid content.

105. The extracted lipid of claim 86, wherein the extracted lipid comprises a total polyunsaturated fatty acid content comprising the total ω6 fatty acid content and the ω3 fatty acid content in total, wherein the total polyunsaturated fatty acid content in the extracted lipid is between about 20% and about 75% of the total fatty acid content.

106. The extracted lipid of claim 86, wherein the extracted plant lipid comprises new ω3 fatty acids in the ω3 fatty acid content in total, wherein the level of new ω3 fatty acids in the extracted lipid is between 9% and about 33% of the total fatty acid content.

107. The extracted lipid of claim 86, wherein at least 80% of the DHA esterified in the form of TAG is in the sn-1 or sn-3 position of the TAG.

108. The extracted lipid of claim 86, wherein the most abundant DHA-containing TAG species in the lipid is DHA/18:3/18:3 (TAG 58:12).

109. The extracted lipid of claim 86, wherein the extracted lipid comprises tri-DHA TAG (TAG 66:18).

110. The extracted lipid of claim 86, which comprises ω6-docosapentaenoic acid (22:5.sup.Δ4, 7, 10, 13, 16) in its total fatty acid content.

111. The extracted lipid of claim 86, wherein the total ω6 fatty acid content in the extracted lipid is less than 20% of the total fatty acid content.

112. The extracted lipid of claim 86, wherein a level of about 1% C20:1 is present in the total fatty acid content in the extracted lipid.

113. Extracted lipid from Brassica seeds, whose total fatty acid content comprises (a) a total monounsaturated fatty acid content which comprises oleic acid, (b) a total saturated fatty acid content which comprises palmitic acid and, if present, myristic acid (C14:0), (c) a total ω6 fatty acid content which comprises linoleic acid (LA), (d) a ω3 fatty acid content, (e) less than 1% myristic acid (C14:0), and (f) less than 4% eicosatrienoic acid (ETrA), wherein (i) a level of DHA of about 3%, about 4', about 5% or about 6% is present in the total fatty acid content of the extracted lipid, wherein at least 70% of the DHA is esterified at the sn-1 or sn-3 position of triacylglycerol (TAG), (ii) a level of palmitic acid between 2% and 16% is present in the total fatty acid content of the extracted lipid, (iii) a level of linoleic acid (LA) between 4% and 35% is present in the total fatty acid content of the extracted lipid, (iv) the total saturated fatty acid content of the extracted lipid is between 4% and 25% of the total fatty acid content, (v) the ratio of the total ω6 fatty acid content to the ω3 fatty acid content in total of the extracted lipid is between 0.1 and 3.0, and (vi) at least 70% of the total fatty acid content of the extracted lipid is esterified in the form of TAG.

Images